Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The supernatant containing antibody product was cleaned up by two rounds of 1.9X SPRI select.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Next, 45 μl of the purified antibody fraction was amplified with a 96 deep well reaction module: 95°C for 3 min; cycled 8 times: 95°C for 20 s, 60°C for 30 s, and 72°C for 20 s; 72°C for 5 min; end at 4°C.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	ADT libraries were purified once more with 1.6X SPRI select.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Full length cDNA, indexed cDNA libraries and antibody libraries were analyzed using the Qubit 4 fluorometer (Thermo Fisher) and Agilent 2100 Bioanalyzer.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The separation of the cDNA and ADT libraries were performed according to the manufacturer’s instructions (10X genomics).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The MACSima™ Imaging System is a fully automated instrument combining liquid handling with widefield microscopy for cyclic immunofluorescence imaging.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	In brief, staining cycles consisted of the following automated steps: immunofluorescent staining, sample washing, multi-field imaging, and signal erasure (photobleaching or REAlease).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Cryosectioned slices on slides were taken out of the -80°C storage and the appropriate MACSWell™ imaging frame was mounted immediately on the slide.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	An appropriate volume of ice-cold 4% PFA solution was added (according to the MACSWell™ imaging frames datasheet) and incubated for 10 minutes at room temperature.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The slide was washed three times with MACSima Running Buffer.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	After washing the appropriate initial sample volume of MACSima Running Buffer was added (according to the MACSWell™ imaging frames datasheet).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Right before the start of the MACSima ™ instrument a DAPI pre-staining was performed: the MACSima Running Buffer was removed from the sample to be analysed and stained for 10 min with a 1:10 dilution of a DAPI staining solution (volume depends on working volume for the different MACSwell™ formats, see datasheet).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The DAPI staining solution was removed and 3 washing steps were performed (MACSima Running Buffer).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Finally, the initial sample volume of MACSima Running Buffer was added.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Details of the antibodies used can be found in the key resources table.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Liver was frozen and sectioned as described above for Visium analysis and liver slices were placed within capture areas on Resolve BioScience slides.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Samples were then sent to Resolve BioSciences on dry ice for analysis.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Upon arrival, tissue sections were thawed and fixed with 4% v/v Formaldehyde (Sigma-Aldrich F8775) in 1x PBS for 30 min at 4 °C.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	After fixation, sections were washed twice in 1x PBS for two min, followed by one min washes in 50% Ethanol and 70% Ethanol at room temperature.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Fixed samples were used for Molecular Cartography™ (100-plex combinatorial single molecule fluorescence in-situ hybridization) according to the manufacturer’s instructions (protocol 3.0; available for download from Resolve’s website to registered users), starting with the aspiration of ethanol and the addition of buffer BST1 (step 6 and 7 of the tissue priming protocol).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Briefly, tissues were primed followed by overnight hybridization of all probes specific for the target genes (see below for probe design details and target list).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Samples were washed the next day to remove excess probes and fluorescently tagged in a two-step color development process.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Regions of interest were imaged as described below and fluorescent signals removed during decolorization.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Color development, imaging and decolorization were repeated for multiple cycles to build a unique combinatorial code for every target gene that was derived from raw images as described below.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The probes for 100 genes were designed using Resolve’s proprietary design algorithm.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Briefly, the probe-design was performed at the gene-level.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	For every targeted gene all full-length protein-coding transcript sequences from the ENSEMBL database were used as design targets if the isoform had the GENCODE annotation tag ‘basic’ (Frankish et al., 2019; Yates et al., 2020).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To speed up the process, the calculation of computationally expensive parts, especially the off-target searches, the selection of probe sequences was not performed randomly, but limited to sequences with high success rates.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To filter highly repetitive regions, the abundance of k-mers was obtained from the background transcriptome using Jellyfish (Marçais and Kingsford, 2011).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Every target sequence was scanned once for all k-mers, and those regions with rare k-mers were preferred as seeds for full probe design.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	A probe candidate was generated by extending a seed sequence until a certain target stability was reached.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	A set of simple rules was applied to discard sequences that were found experimentally to cause problems.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	After these fast screens, every kept probe candidate was mapped to the background transcriptome using ThermonucleotideBLAST (Gans and Wolinsky, 2008) and probes with stable off-target hits were discarded.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Specific probes were then scored based on the number of on-target matches (isoforms), which were weighted by their associated APPRIS level (Rodriguez et al., 2018), favoring principal isoforms over others.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	A bonus was added if the binding-site was inside the protein-coding region.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	From the pool of accepted probes, the final set was composed by greedily picking the highest scoring probes.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Probe details are included in the key resources table.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Samples were imaged on a Zeiss Celldiscoverer 7, using the 50x Plan Apochromat water immersion objective with an NA of 1.2 and the 0.5x magnification changer, resulting in a 25x final magnification.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Standard CD7 LED excitation light source, filters, and dichroic mirrors were used together with customized emission filters optimized for detecting specific signals.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Excitation time per image was 1000 ms for each channel (DAPI was 20 ms).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	A z-stack was taken at each region with a distance per z-slice according to the Nyquist-Shannon sampling theorem.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The custom CD7 CMOS camera (Zeiss Axiocam Mono 712, 3.45 μm pixel size) was used.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	For each region, a z-stack per fluorescent color (two colors) was imaged per imaging round.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	A total of 8 imaging rounds were done for each position, resulting in 16 z-stacks per region.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The completely automated imaging process per round (including water immersion generation and precise relocation of regions to image in all three dimensions) was realized by a custom python script using the scripting API of the Zeiss ZEN software (Open application development).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The algorithms for spot segmentation were written in Java and are based on the ImageJ library functionalities.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Only the iterative closest point algorithm is written in C++ based on the libpointmatcher library (https://github.com/ethz-asl/libpointmatcher).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	As a first step all images were corrected for background fluorescence.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	A target value for the allowed number of maxima was determined based upon the area of the slice in μm multiplied by the factor 0.5.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This factor was empirically optimized.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The brightest maxima per plane were determined, based upon an empirically optimized threshold.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The number and location of the respective maxima was stored.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This procedure was done for every image slice independently.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Maxima that did not have a neighboring maximum in an adjacent slice (called z-group) were excluded.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The resulting maxima list was further filtered in an iterative loop by adjusting the allowed thresholds for (Babs-Bback) and (Bperi-Bback) to reach a feature target value (Babs: absolute brightness, Bback: local background, Bperi: background of periphery within 1 pixel).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This feature target values were based upon the volume of the 3D-image.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Only maxima still in a z-group of at least 2 after filtering were passing the filter step.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Each z-group was counted as one hit.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The members of the z-groups with the highest absolute brightness were used as features and written to a file.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	They resemble a 3D-point cloud.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To align the raw data images from different imaging rounds, images had to be corrected.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To do so the extracted feature point clouds were used to find the transformation matrices.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	For this purpose, an iterative closest point cloud algorithm was used to minimize the error between two point-clouds.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The point clouds of each round were aligned to the point cloud of round one (reference point cloud).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The corresponding point clouds were stored for downstream processes.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Based upon the transformation matrices the corresponding images were processed by a rigid transformation using trilinear interpolation.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The aligned images were used to create a profile for each pixel consisting of 16 values (16 images from two color channels in 8 imaging rounds).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The pixel profiles were filtered for variance from zero normalized by total brightness of all pixels in the profile.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Matched pixel profiles with the highest score were assigned as an ID to the pixel.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Pixels with neighbors having the same ID were grouped.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The pixel groups were filtered by group size, number of direct adjacent pixels in group, number of dimensions with size of two pixels.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The local 3D-maxima of the groups were determined as potential final transcript locations.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Maxima were filtered by number of maxima in the raw data images where a maximum was expected.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Remaining maxima were further evaluated by the fit to the corresponding code.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The remaining maxima were written to the results file and considered to resemble transcripts of the corresponding gene.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The ratio of signals matching to codes used in the experiment and signals matching to codes not used in the experiment were used as estimation for specificity (false positives).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Final image analysis was performed in ImageJ using genexyz Polylux tool plugin from Resolve BioSciences to examine specific Molecular Cartography™ signals.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	40000-160000 cells of interest from livers of the different species were FACS-purified and pelleted by centrifugation at 400g for 5 mins.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To ensure sufficient numbers of all cell types were present in our analyses, depending on the sample distinct populations of cells including Live CD45, Live CD45, Hepatocytes, Myeloid cells and Stromal cells were FACS-purified.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	When CITE-seq was to be performed, cells were then stained with 2.4G2 antibody to block Fc receptors and CITE-seq antibodies for 20mins at 4°C, before being washed in excess PBS with 2% FCS and 2mM EDTA.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Antibody details are included in the key resources table.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	40000-100000 nuclei were also FACS-purified based on DAPI expression.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	These were sorted into BSA coated tubes and pelleted by centrifuging for 3 mins at 400g and 5 mins at 600g sequentially.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Cells/Nuclei were then resuspended in PBS with 0.04%BSA at ∼1000 cells/ml.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Cell suspensions (target recovery of 8000-10000 cells) were loaded on a GemCode Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead-in-Emulsions (GEMs).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Single-cell RNA-Seq libraries were prepared using GemCode Single-Cell 3ʹGel Bead and Library Kit (10x Genomics, V2 and V3 technology) according to the manufacturer’s instructions.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Briefly, GEM-RT was performed in a 96-Deep Well Reaction Module: 55°C for 45min, 85°C for 5 min; end at 4°C.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	After RT, GEMs were broken down and the cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, 37002D) and SPRIselect Reagent Kit (SPRI; Beckman Coulter; B23318).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	cDNA was amplified with 96-Deep Well Reaction Module: 98°C for 3 min; cycled 12 times : 98°C for 15s, 67°C for 20 s, and 72°C for 1 min; 72°C for 1 min; end at 4°C.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Amplified cDNA product was cleaned up with SPRIselect Reagent Kit prior to enzymatic fragmentation.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Indexed sequencing libraries were generated using the reagents in the GemCode Single-Cell 3ʹ Library Kit with the following intermediates: (1) end repair; (2) A-tailing; (3) adapter ligation; (4) post-ligation SPRIselect cleanup and (5) sample index PCR.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Pre-fragmentation and post-sample index PCR samples were analyzed using the Agilent 2100 Bioanalyzer.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	RNA was extracted from 10000 sorted cells (gated using strategies shown) from livers of C57BL/6 mice using a RNeasy Plus micro kit (QIAGEN).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Sensifast cDNA synthesis kit (Bioline) was used to transcribe total RNA to cDNA.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Real-time RT-PCR using SensiFast SYBR No-Rox kit (Bioline) was performed to determine gene expression, therefore a PCR amplification and detection instrument LightCycler 480 (Roche) was used.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Gene expression was normalized to β-actin gene expression.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Primers used in the study can be found in the key resources table.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	sc/snRNA-seq libraries were loaded on an Illumina HiSeq or Illumina NovaSeq 6000 with sequencing settings recommended by 10X Genomics (26/8/0/98 – 2.1pM loading concentration, ADT and cDNA libraries were pooled in a 25:75 ratio).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Visium sequencing libraries were loaded on an Illumina NovaSeq 6000 with sequencing settings recommended by 10X Genomics (28/10/10/75 – 2.1pM loading concentration).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Sequencing was performed at the VIB Nucleomics Core (VIB, Leuven).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The supernatant containing antibody product was cleaned up by two rounds of 1.9X SPRI select.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Next, 45 μl of the purified antibody fraction was amplified with a 96 deep well reaction module: 95°C for 3 min; cycled 8 times: 95°C for 20 s, 60°C for 30 s, and 72°C for 20 s; 72°C for 5 min; end at 4°C.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

ADT libraries were purified once more with 1.6X SPRI select.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Full length cDNA, indexed cDNA libraries and antibody libraries were analyzed using the Qubit 4 fluorometer (Thermo Fisher) and Agilent 2100 Bioanalyzer.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The separation of the cDNA and ADT libraries were performed according to the manufacturer’s instructions (10X genomics).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The MACSima™ Imaging System is a fully automated instrument combining liquid handling with widefield microscopy for cyclic immunofluorescence imaging.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

In brief, staining cycles consisted of the following automated steps: immunofluorescent staining, sample washing, multi-field imaging, and signal erasure (photobleaching or REAlease).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Cryosectioned slices on slides were taken out of the -80°C storage and the appropriate MACSWell™ imaging frame was mounted immediately on the slide.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

An appropriate volume of ice-cold 4% PFA solution was added (according to the MACSWell™ imaging frames datasheet) and incubated for 10 minutes at room temperature.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The slide was washed three times with MACSima Running Buffer.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

After washing the appropriate initial sample volume of MACSima Running Buffer was added (according to the MACSWell™ imaging frames datasheet).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Right before the start of the MACSima ™ instrument a DAPI pre-staining was performed: the MACSima Running Buffer was removed from the sample to be analysed and stained for 10 min with a 1:10 dilution of a DAPI staining solution (volume depends on working volume for the different MACSwell™ formats, see datasheet).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The DAPI staining solution was removed and 3 washing steps were performed (MACSima Running Buffer).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Finally, the initial sample volume of MACSima Running Buffer was added.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Details of the antibodies used can be found in the key resources table.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Liver was frozen and sectioned as described above for Visium analysis and liver slices were placed within capture areas on Resolve BioScience slides.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Samples were then sent to Resolve BioSciences on dry ice for analysis.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Upon arrival, tissue sections were thawed and fixed with 4% v/v Formaldehyde (Sigma-Aldrich F8775) in 1x PBS for 30 min at 4 °C.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

After fixation, sections were washed twice in 1x PBS for two min, followed by one min washes in 50% Ethanol and 70% Ethanol at room temperature.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Fixed samples were used for Molecular Cartography™ (100-plex combinatorial single molecule fluorescence in-situ hybridization) according to the manufacturer’s instructions (protocol 3.0; available for download from Resolve’s website to registered users), starting with the aspiration of ethanol and the addition of buffer BST1 (step 6 and 7 of the tissue priming protocol).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Briefly, tissues were primed followed by overnight hybridization of all probes specific for the target genes (see below for probe design details and target list).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Samples were washed the next day to remove excess probes and fluorescently tagged in a two-step color development process.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Regions of interest were imaged as described below and fluorescent signals removed during decolorization.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Color development, imaging and decolorization were repeated for multiple cycles to build a unique combinatorial code for every target gene that was derived from raw images as described below.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The probes for 100 genes were designed using Resolve’s proprietary design algorithm.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Briefly, the probe-design was performed at the gene-level.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

For every targeted gene all full-length protein-coding transcript sequences from the ENSEMBL database were used as design targets if the isoform had the GENCODE annotation tag ‘basic’ (Frankish et al., 2019; Yates et al., 2020).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To speed up the process, the calculation of computationally expensive parts, especially the off-target searches, the selection of probe sequences was not performed randomly, but limited to sequences with high success rates.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To filter highly repetitive regions, the abundance of k-mers was obtained from the background transcriptome using Jellyfish (Marçais and Kingsford, 2011).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Every target sequence was scanned once for all k-mers, and those regions with rare k-mers were preferred as seeds for full probe design.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

A probe candidate was generated by extending a seed sequence until a certain target stability was reached.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

A set of simple rules was applied to discard sequences that were found experimentally to cause problems.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

After these fast screens, every kept probe candidate was mapped to the background transcriptome using ThermonucleotideBLAST (Gans and Wolinsky, 2008) and probes with stable off-target hits were discarded.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Specific probes were then scored based on the number of on-target matches (isoforms), which were weighted by their associated APPRIS level (Rodriguez et al., 2018), favoring principal isoforms over others.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

A bonus was added if the binding-site was inside the protein-coding region.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

From the pool of accepted probes, the final set was composed by greedily picking the highest scoring probes.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Probe details are included in the key resources table.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Samples were imaged on a Zeiss Celldiscoverer 7, using the 50x Plan Apochromat water immersion objective with an NA of 1.2 and the 0.5x magnification changer, resulting in a 25x final magnification.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Standard CD7 LED excitation light source, filters, and dichroic mirrors were used together with customized emission filters optimized for detecting specific signals.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Excitation time per image was 1000 ms for each channel (DAPI was 20 ms).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

A z-stack was taken at each region with a distance per z-slice according to the Nyquist-Shannon sampling theorem.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The custom CD7 CMOS camera (Zeiss Axiocam Mono 712, 3.45 μm pixel size) was used.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

For each region, a z-stack per fluorescent color (two colors) was imaged per imaging round.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

A total of 8 imaging rounds were done for each position, resulting in 16 z-stacks per region.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The completely automated imaging process per round (including water immersion generation and precise relocation of regions to image in all three dimensions) was realized by a custom python script using the scripting API of the Zeiss ZEN software (Open application development).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The algorithms for spot segmentation were written in Java and are based on the ImageJ library functionalities.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Only the iterative closest point algorithm is written in C++ based on the libpointmatcher library (https://github.com/ethz-asl/libpointmatcher).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

As a first step all images were corrected for background fluorescence.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

A target value for the allowed number of maxima was determined based upon the area of the slice in μm multiplied by the factor 0.5.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This factor was empirically optimized.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The brightest maxima per plane were determined, based upon an empirically optimized threshold.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The number and location of the respective maxima was stored.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This procedure was done for every image slice independently.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Maxima that did not have a neighboring maximum in an adjacent slice (called z-group) were excluded.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The resulting maxima list was further filtered in an iterative loop by adjusting the allowed thresholds for (Babs-Bback) and (Bperi-Bback) to reach a feature target value (Babs: absolute brightness, Bback: local background, Bperi: background of periphery within 1 pixel).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This feature target values were based upon the volume of the 3D-image.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Only maxima still in a z-group of at least 2 after filtering were passing the filter step.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Each z-group was counted as one hit.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The members of the z-groups with the highest absolute brightness were used as features and written to a file.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

They resemble a 3D-point cloud.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To align the raw data images from different imaging rounds, images had to be corrected.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To do so the extracted feature point clouds were used to find the transformation matrices.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

For this purpose, an iterative closest point cloud algorithm was used to minimize the error between two point-clouds.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The point clouds of each round were aligned to the point cloud of round one (reference point cloud).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The corresponding point clouds were stored for downstream processes.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Based upon the transformation matrices the corresponding images were processed by a rigid transformation using trilinear interpolation.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The aligned images were used to create a profile for each pixel consisting of 16 values (16 images from two color channels in 8 imaging rounds).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The pixel profiles were filtered for variance from zero normalized by total brightness of all pixels in the profile.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Matched pixel profiles with the highest score were assigned as an ID to the pixel.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Pixels with neighbors having the same ID were grouped.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The pixel groups were filtered by group size, number of direct adjacent pixels in group, number of dimensions with size of two pixels.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The local 3D-maxima of the groups were determined as potential final transcript locations.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Maxima were filtered by number of maxima in the raw data images where a maximum was expected.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Remaining maxima were further evaluated by the fit to the corresponding code.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The remaining maxima were written to the results file and considered to resemble transcripts of the corresponding gene.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The ratio of signals matching to codes used in the experiment and signals matching to codes not used in the experiment were used as estimation for specificity (false positives).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Final image analysis was performed in ImageJ using genexyz Polylux tool plugin from Resolve BioSciences to examine specific Molecular Cartography™ signals.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

40000-160000 cells of interest from livers of the different species were FACS-purified and pelleted by centrifugation at 400g for 5 mins.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To ensure sufficient numbers of all cell types were present in our analyses, depending on the sample distinct populations of cells including Live CD45, Live CD45, Hepatocytes, Myeloid cells and Stromal cells were FACS-purified.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

When CITE-seq was to be performed, cells were then stained with 2.4G2 antibody to block Fc receptors and CITE-seq antibodies for 20mins at 4°C, before being washed in excess PBS with 2% FCS and 2mM EDTA.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Antibody details are included in the key resources table.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

40000-100000 nuclei were also FACS-purified based on DAPI expression.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

These were sorted into BSA coated tubes and pelleted by centrifuging for 3 mins at 400g and 5 mins at 600g sequentially.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Cells/Nuclei were then resuspended in PBS with 0.04%BSA at ∼1000 cells/ml.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Cell suspensions (target recovery of 8000-10000 cells) were loaded on a GemCode Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead-in-Emulsions (GEMs).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Single-cell RNA-Seq libraries were prepared using GemCode Single-Cell 3ʹGel Bead and Library Kit (10x Genomics, V2 and V3 technology) according to the manufacturer’s instructions.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Briefly, GEM-RT was performed in a 96-Deep Well Reaction Module: 55°C for 45min, 85°C for 5 min; end at 4°C.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

After RT, GEMs were broken down and the cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, 37002D) and SPRIselect Reagent Kit (SPRI; Beckman Coulter; B23318).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

cDNA was amplified with 96-Deep Well Reaction Module: 98°C for 3 min; cycled 12 times : 98°C for 15s, 67°C for 20 s, and 72°C for 1 min; 72°C for 1 min; end at 4°C.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Amplified cDNA product was cleaned up with SPRIselect Reagent Kit prior to enzymatic fragmentation.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Indexed sequencing libraries were generated using the reagents in the GemCode Single-Cell 3ʹ Library Kit with the following intermediates: (1) end repair; (2) A-tailing; (3) adapter ligation; (4) post-ligation SPRIselect cleanup and (5) sample index PCR.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Pre-fragmentation and post-sample index PCR samples were analyzed using the Agilent 2100 Bioanalyzer.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

RNA was extracted from 10000 sorted cells (gated using strategies shown) from livers of C57BL/6 mice using a RNeasy Plus micro kit (QIAGEN).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Sensifast cDNA synthesis kit (Bioline) was used to transcribe total RNA to cDNA.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Real-time RT-PCR using SensiFast SYBR No-Rox kit (Bioline) was performed to determine gene expression, therefore a PCR amplification and detection instrument LightCycler 480 (Roche) was used.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Gene expression was normalized to β-actin gene expression.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Primers used in the study can be found in the key resources table.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

sc/snRNA-seq libraries were loaded on an Illumina HiSeq or Illumina NovaSeq 6000 with sequencing settings recommended by 10X Genomics (26/8/0/98 – 2.1pM loading concentration, ADT and cDNA libraries were pooled in a 25:75 ratio).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Visium sequencing libraries were loaded on an Illumina NovaSeq 6000 with sequencing settings recommended by 10X Genomics (28/10/10/75 – 2.1pM loading concentration).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Sequencing was performed at the VIB Nucleomics Core (VIB, Leuven).