cel_seq/CEL-Seq_protocol.docling_text.txt

# docling text extraction
source_pdf: CEL-Seq_protocol.pdf
source_path: /Users/seqmachines/playground/protocols-test/scg-v1-upload/protocols/cel_seq/CEL-Seq_protocol.pdf
extraction: Docling: PdfPipelineOptions(do_ocr=False); assembled markdown plus Docling native PDF layout prediction text cells sorted by page/y/x

## Docling assembled markdown

## CEL-Seq Protocol

## By Tamar Hashimshony, Technion July 16 th , 2012

## Reagents:

LoBind tubes - 0.5 ml - Eppendorf 022431005 Ultra pure RNase free water Ethanol Bioanalyzer kits - Agilent RNA pico kit (5067-1513), high sensitivity DNA kit (5067-4626) Qubit reagents: dsDNA HS Assay - invitrogen Q32851 or Q32854 For RNA amplification: ERCC RNA spike-in mix - Ambion 4456740 MessageAmpII kit - Ambion AM1751 Optional: extra columns for cDNA/aRNA purification - Ambion10066G Fragmentation buffer: 200mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc Fragmentation stop buffer: 0.5 M EDTA For Library preparation: Antarctic phosphatase - NEB M0289 RNase OUT - invitrogen 100000840 PNK - NEB M0201 Superscript II - invitrogen 18064-014 RNeasy MinElute kit - Qiagen 74204 T4 RNA ligase 2, truncated - NEB M0242 AMPure XP beads - Beckman Coulter A63880 TruSeq small RNA sample prep kit - Illumina RS-200-0012 (or -0024, -0036, -0048) Optional (to supplement Illumina's Small RNA kit): ATP - NEB P0756 Phusion® High-Fidelity PCR Master Mix with HF Buffer - NEB M0531 RNA RT primer, RNA PCR primers (sequences available from Illumina)

## Equipment:

Thermocycler with lid with adjustable temperature (one that can also fit 0.5 ml PCR tubes is convenient) Speed vac Oven Heat block Magnetic stand (for 0.5 ml tubes) Qubit® Fluorometer - invitrogen Bioanalyzer - Agilent

## Primers:

CEL-Seq primer design: The RT primer was designed with an anchored polyT, a unique barcode, the 5' Illumina adapter (as used in the Illumina small RNA kit) and a T7 promoter. The barcodes were of length eight and designed in groups of four, such that the first five nucleotides will have equal representation first four nucleotides read in the Illumina platform. The barcodes were designed such that each pair is different by at least two nucleotides, so that a single sequencing error will not produce the wrong

of all four nucleotides to allow for template generation and crosstalk corrections which are based on the barcode. Primers are desalted, stock solution 1 μg/μl, working concentration 25ng/μl. #1:  CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCATCACGCTTTTTTTTTTTTTTTTTTTTTTTTV #2: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGTCGTCGCTTTTTTTTTTTTTTTTTTTTTTTTV #3: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCACGACCGCTTTTTTTTTTTTTTTTTTTTTTTTV #4: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTGATGCGCTTTTTTTTTTTTTTTTTTTTTTTTV #5: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCATCAATCTTTTTTTTTTTTTTTTTTTTTTTTV #6: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGTCGTATCTTTTTTTTTTTTTTTTTTTTTTTTV #7: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCACGACATCTTTTTTTTTTTTTTTTTTTTTTTTV #8: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTGATGATCTTTTTTTTTTTTTTTTTTTTTTTTV #9: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCATCATCCTTTTTTTTTTTTTTTTTTTTTTTTV #10: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGTCGTTCCTTTTTTTTTTTTTTTTTTTTTTTTV #11: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCACGACTCCTTTTTTTTTTTTTTTTTTTTTTTTV #12: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTGATGTCCTTTTTTTTTTTTTTTTTTTTTTTTV #13: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCATCAGAATTTTTTTTTTTTTTTTTTTTTTTTV #14: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGTCGTGAATTTTTTTTTTTTTTTTTTTTTTTTV #15: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCACGACGAATTTTTTTTTTTTTTTTTTTTTTTTV #16: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTGATGGAATTTTTTTTTTTTTTTTTTTTTTTTV #17: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTCACACGCTTTTTTTTTTTTTTTTTTTTTTTTV #18: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCGTGTCGCTTTTTTTTTTTTTTTTTTTTTTTTV #19: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGACACCGCTTTTTTTTTTTTTTTTTTTTTTTTV #20: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCATGTGCGCTTTTTTTTTTTTTTTTTTTTTTTTV #21: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTCACATCATTTTTTTTTTTTTTTTTTTTTTTTV #22: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCGTGTTCATTTTTTTTTTTTTTTTTTTTTTTTV #23: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGACACTCATTTTTTTTTTTTTTTTTTTTTTTTV #24: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCATGTGTCATTTTTTTTTTTTTTTTTTTTTTTTV #25: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTCACAGAGTTTTTTTTTTTTTTTTTTTTTTTTV #26: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCGTGTGAGTTTTTTTTTTTTTTTTTTTTTTTTV #27:CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGACACGAGTTTTTTTTTTTTTTTTTTTTTTTTV #28: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCATGTGGAGTTTTTTTTTTTTTTTTTTTTTTTTV #29: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCTAACCGCTTTTTTTTTTTTTTTTTTTTTTTTV #30: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGCTTGCGCTTTTTTTTTTTTTTTTTTTTTTTTV #31: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCAGCCACGCTTTTTTTTTTTTTTTTTTTTTTTTV #32: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTAGGTCGCTTTTTTTTTTTTTTTTTTTTTTTTV #33: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCTAACTCATTTTTTTTTTTTTTTTTTTTTTTTV #34: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGCTTGTCATTTTTTTTTTTTTTTTTTTTTTTTV #35: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCAGCCATCATTTTTTTTTTTTTTTTTTTTTTTTV #36: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTAGGTTCATTTTTTTTTTTTTTTTTTTTTTTTV #37: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCTAACGAGTTTTTTTTTTTTTTTTTTTTTTTTV #38: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGCTTGGAGTTTTTTTTTTTTTTTTTTTTTTTTV #39:CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCAGCCAGAGTTTTTTTTTTTTTTTTTTTTTTTTV #40: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTAGGTGAGTTTTTTTTTTTTTTTTTTTTTTTTV

## Single cell isolation :

Individual cells (so far we've worked with C. elegans blastomeres or trypsinised tissue culture cells) are transferred with a micro-pipette into a 0.5µl drop of appropriate buffer (egg salts or PBS) placed on the cap of a 0.5 ml LoBind Eppendorf tube. Location of cell should be marked. Excess liquid is aspirated off, and tube is frozen in liquid nitrogen. Samples are stored at -80°C.

## RNA Amplification:

## Prepare primer mix (for each different primer used):

Primer (25ng/μl)

1μl

ERCC Spike-in          Xμl

water

Yμl

6μl

Spike-in dilution should be appropriate for sample size - see protocol of ERCC RNA spike in mix. For single cells we add 1ul of spike-in at 1:500,000 dilution.

## Breaking cell open and annealing with primer:

-  Add 1.2μl primer mix to marked location of single cell on cap of tube (Keep cell frozen until adding the primer mix, handle up to 12 cells in parallel).
-  Incubate 5 min. at 70 o C (with lid of thermal cycler set to 70 o C).
-  Brief spin down.
-  Incubate for an additional 5 min. at 70 o C.
-  Move immediately to ice.
-  Spin at maximal speed for a few seconds to collect as many droplets as possible before next step, and then return to ice.

## RT reaction (Ambion kit)

-  Add 0.8μl of the following mix to each reaction:
-  Incubate 2hr at 42 o C ( in hybridization oven)

First Strand buffer

0.2μl

dNTP

0.4μl

RNase Inhibitor

0.1μl

ArrayScript

0.1μl

## Second strand reaction (Ambion kit):

-  Move previous step to ice so it cools below 16 o C.
-  Add 8uL of the following mix to each reaction tube:

| DDW                | 6.3μl   |
|--------------------|---------|
| Second strand buf. | 1μl     |
| dNTP               | 0.4μl   |
| DNA Pol            | 0.2μl   |
| RNaseH             | 0.1μl   |

Flick and spin samples (at maximal speed for a few seconds).

-  Incubate at 16 o C for 2hr (in thermal cycler with unheated or open lid).

## cDNA cleanup and speedvac:

-  Pool all cells that are to go to same IVT.  Should have ~10μl from each cell.
-  Adjust volume to 100μl with nuclease free water.  If more than 10 cells in a pool, just add all together.
-  Add 250μl cDNA binding buffer to each 100ul sample (if total sample volume exceeds 100uL, adjust cDNA binding buffer volume according to sample volume). Load onto Ambion cDNA cleanup column. Volume of up to 24 pooled samples can be loaded. If more than 24 samples are pooled, after spin load remaining volume and spin again.
-  Spin 1 min at 10,000g to bind cDNA, discard flow through (repeat if more than 24 samples are pooled).
-  Add 500μl wash buffer, spin as above, discard flow through.
-  Spin for an additional minute to dry.
-  Transfer column to clean round bottom 2 ml tube.
-  Elute by adding 9μl warm water (55 o C), incubating for 2 minutes at room temperature and spinning 1.5 min at 10,000g.
-  Repeat elution.
-  Adjust volume to 6.4μl by drying in a speedvac (~8 min).

Stopping point: Samples can be kept at -20 o C

## IVT (Ambion kit):

-  Prepare the following mix and add 9.6μl per tube.

A 1.6μl G 1.6μl C 1.6μl U 1.6μl 10xT7 buffer 1.6μl T7 enzyme 1.6μl

-  Incubate in a thermal cycler at 37 o C for 13 hrs, with lid at 70 o C.  Set cycler to go to 4 o C at end of incubation. aRNA (amplified RNA) is stable for at least several hours.

## RNA fragmentation and cleanup:

-  Mix the following on ice:

aRNA 16μl

Fragmentation buffer 4μl

-  Incubate for 3 min. at 94 o C.
-  Immediately move to ice and add 2μl fragmentation stop buffer.
-  Adjust volume to 30ul by adding 8μl water.
-  Add 105μl aRNA binding buffer, followed by 75μl EtOH, immediately mix by pipetting 3-4 times and load onto spin-column. Bind sample by immediately spinning for 1 min at 10,000g, discard flow through. (If cleaning more than one reaction, this step should be done for each sample separately)
-  Add 500μl wash buffer (samples can wait at this step until binding of all samples is complete).
-  Spin as above, discard flow through.
-  Spin for an additional minute to dry.
-  Transfer column to clean tube.
-  Elute by adding 10μl warm water (55 o C), incubating for 2 minutes at room temperature and spinning 1.5 min at 10,000 g.
-  Repeat elution.

Stopping point: Samples can be kept at -80

## Check aRNA amount and quality:

-  Load 1μl onto Bioanalyzer RNA pico chip after heating an aliquot of the sample to 70 o for 2 min.
-  When starting the IVT with ~0.5ng total RNA, the expected yield is 500-1000 pg/μl. Size distribution should peak at ~500 bp (See Bioanalyzer plot for example).

<!-- image -->

o C

## Library preparation:

Protocol designed for 5-10ng amplified RNA, although as little as 1-2ng can be used, but then additional PCR cycles are required. Sample volume should be adjusted to 16μl, either by adding water or drying down in a speedvac, depending on RNA concentration. IVTs can be pooled at this point if there is no overlap in barcodes used.

## Phosphatase treatment:

To 16μl of fragmented aRNA in a 0.7ml PCR tube add 4μl of the following mix:

-  10X phospatase buffer 2μl

-  Antarctic phosphatase 1μl

-  RNaseOUT 1μl

Incubate in a thermal cycler with the following protocol:

-  37°C for 30 minutes
-  65°C for 5 minutes
-  4°C indefinite hold

## PNK treatment:

To the 0.7 ml PCR tube from the previous step add 30μl of the following mix:

-  nuclease-free H2O 17μl

-  10X phosphatase buffer 5μl

-  ATP (10mM, from Illumina kit) 5μl

-  RNaseOUT 1μl

-  PNK 2μl

Incubate in a thermal cycler at 37°C for 60 minutes then 4°C hold.

## Column cleanup of phosphatase and PNK treated aRNA (RNeasy kit):

-  Adjust the volume to 100μl (add 50μL) using nuclease free water.
-  Add 350μl of RLT buffer and mix well.
-  Add 250μl EtOH, mix well by pipetting, and transfer sample to an RNeasy spin column.
-  Spin 15 sec. at 8,000 g.
-  Transfer column to new collection tube, and add 500μl Buffer RPE.
-  Spin 15 sec. at 8,000 g.
-  Discard flow-through, and add 500 ml 80% EtOH.
-  Spin 2 min. at 8,000 g.
-  Transfer column to new collection tube, open lid of column, and spin 5 min at full speed.
-  Transfer column to new collection tube, and elute with 14μl nuclease free H2O, spinning at full speed for 1 minute.
-  Dry down the sample using a speedvac to 5μl. (approx. 7 minutes)

## Ligate 3' adapter:

Dilute 3' adapter (RA3, from Illumina kit) 5 fold.

To 5μl phosphatase and PNK treated RNA add 1μl of the diluted 3' adaptor.

Incubate at 70°C for 2 minutes and then immediately place the tube on ice to prevent secondary structure formation.

## Add 4μl of the following mix:

-  5X HM Ligation Buffer (HML, Illumina kit)

2μL

- 

- RNase Inhibitor (Illumina kit) 1μL

- 

- T4 RNA Ligase 2, truncated 1μL

Incubate the tube on the pre-heated thermal cycler at 28°C for 1 hour (with unheated or open lid).

With the reaction tube remaining on the thermal cycler, add 1μl Stop Solution (STP, Illumina kit) and gently pipette the entire volume up and down 6-8 times to mix thoroughly. Continue to incubate the reaction tube on the thermal cycler at 28°C for 15 minutes, and then place the tube on ice.

Add 3μL nuclease free water.

## Reverse transcription reaction:

Dilute dNTPs (from Illumina kit) two fold with nuclease free water (prepare at least 1μl per sample)

Combine the following in a PCR tube (the remaining 3' adapter-ligated RNA may be stored at -80°C):

-  Adapter-ligated RNA 6μL

-  RNA RT Primer (RTP, from Illumina kit)   1μL

Incubate the tube at 70°C for 2 minutes and then immediately place the tube on ice.

Add 5.5μl of the following mix:

-  5X First Strand Buffer

2μL

-  12.5 mM dNTP mix (diluted dNTP)

0.5μL

-  100 mM DTT 1μL

- 

- RNase Inhibitor (Illumina kit) 1μL

-  SuperScript II Reverse Transcriptase

1μL

Incubate the tube in the pre-heated thermal cycler at 50°C for 1 hour and then place the tube on ice.

## PCR amplification:

To each reverse transcription reaction add 35.5μl of the following mix:

-  Ultra Pure Water

8.5μL

-  PCR mix (PML, from Illumina kit))

25μL

-  RNA PCR Primer (RP1, from Illumina kit)

2μL

To each reaction add 2μl of a uniquely indexed RNA PCR Primer (RPIX, from Illumina kit)

Amplify the tube in the thermal cycler using the following PCR cycling conditions:

-  30 seconds at 98°C
-  12 cycles of:
-  10 seconds at 98°C
-  30 seconds at 60°C
-  30 seconds at 72°C
-  10 minutes at 72°C
-  Hold at 4°C

Can go up to 15 cycles if necessary, or down to 11 if starting with the full 10ng.

Stopping point: samples can be kept at -20 o C.

## Bead Cleanup of PCR products - Repeat 1:

-  Prewarm beads to room temperature.
-  Vortex AMPure XP Beads until well dispersed, then add 50μl to the 50μl PCR reaction. Mix entire volume up ten times to mix thoroughly.
-  Incubate at room temperature for 15 min.
-  Place on magnetic stand for at least 5 min, until liquid appears clear.
-  Remove and discard 95μl of the supernatant.
-  Add 200μl freshly prepared 80% EtOH.
-  Incubate at least 30 seconds, then remove and discard supernatant without disturbing beads.
-  Add 200μl freshly prepared 80% EtOH
-  Incubate at least 30 seconds, then remove and discard supernatant without disturbing beads.
-  Air dry beads for 15 min, or until completely dry.
-  Resuspend with 32.5μl Resuspension Buffer (from Illumina kit). Pipette entire volume up and down ten times to mix thoroughly.
-  Incubate at room temperature for 2 min.
-  Place on magnetic stand for 5 min, until liquid appears clear.
-  Transfer 30μl of supernatant to new tube.

## Bead Cleanup of PCR products - Repeat 2:

Repeat as above, but adding 39μl beads and eluting in 12.5μl resuspension buffer at the end, transferring 10μl to a new tube.

## Check library amount and quality:

Check concentration of DNA by Qubit, 1μl should be enough to measure using the high sensitivity reagent; expected concentration is at least ~1ng/μl.

Run 1μl of each sample on Bioanalyzer using a high sensitivity DNA chip to see size distribution. Expected peak at 300-400bp (See Bioanalyzer plot for example).

<!-- image -->

Concentration to be loaded for sequencing should be calibrated by the sequencing facility. For us, 5pM on Hi-Seq v.1 reagents gave good cluster density.

## Docling layout prediction text cells

### Page 1

CEL-Seq Protocol
By Tamar Hashimshony, Technion
July 16 th , 2012
Reagents:
LoBind tubes - 0.5 ml - Eppendorf 022431005
Ultra pure RNase free water
Ethanol
Bioanalyzer kits - Agilent RNA pico kit (5067-1513), high sensitivity DNA kit (5067-4626)
Qubit reagents: dsDNA HS Assay - invitrogen Q32851 or Q32854
For RNA amplification:
ERCC RNA spike-in mix - Ambion 4456740
MessageAmpII kit - Ambion AM1751
Optional: extra columns for cDNA/aRNA purification - Ambion10066G
Fragmentation buffer: 200mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc
Fragmentation stop buffer: 0.5 M EDTA
For Library preparation:
Antarctic phosphatase - NEB M0289
RNase OUT - invitrogen 100000840
PNK - NEB M0201
Superscript II - invitrogen 18064-014
RNeasy MinElute kit - Qiagen 74204
T4 RNA ligase 2, truncated - NEB M0242
AMPure XP beads - Beckman Coulter A63880
TruSeq small RNA sample prep kit - Illumina RS-200-0012 (or -0024, -0036, -0048)
Optional (to supplement Illumina's Small RNA kit):
ATP - NEB P0756
Phusion® High-Fidelity PCR Master Mix with HF Buffer - NEB M0531
RNA RT primer, RNA PCR primers (sequences available from Illumina)
Equipment:
Thermocycler with lid with adjustable temperature (one that can also fit 0.5 ml PCR tubes is convenient)
Speed vac
Oven
Heat block
Magnetic stand (for 0.5 ml tubes)
Qubit® Fluorometer - invitrogen
Bioanalyzer - Agilent

### Page 2

Primers:
CEL-Seq primer design: The RT primer was designed with an anchored polyT, a unique barcode, the 5'
Illumina adapter (as used in the Illumina small RNA kit) and a T7 promoter. The barcodes were of length
eight and designed in groups of four, such that the first five nucleotides will have equal representation
of all four nucleotides to allow for template generation and crosstalk corrections which are based on the
first four nucleotides read in the Illumina platform. The barcodes were designed such that each pair is
different by at least two nucleotides, so that a single sequencing error will not produce the wrong
barcode. Primers are desalted, stock solution 1 μg/μl, working concentration 25ng/μl.
#1:  CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCATCACGCTTTTTTTTTTTTTTTTTTTTTTTTV
#2: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGTCGTCGCTTTTTTTTTTTTTTTTTTTTTTTTV
#3: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCACGACCGCTTTTTTTTTTTTTTTTTTTTTTTTV
#4: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTGATGCGCTTTTTTTTTTTTTTTTTTTTTTTTV
#5: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCATCAATCTTTTTTTTTTTTTTTTTTTTTTTTV
#6: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGTCGTATCTTTTTTTTTTTTTTTTTTTTTTTTV
#7: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCACGACATCTTTTTTTTTTTTTTTTTTTTTTTTV
#8: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTGATGATCTTTTTTTTTTTTTTTTTTTTTTTTV
#9: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCATCATCCTTTTTTTTTTTTTTTTTTTTTTTTV
#10: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGTCGTTCCTTTTTTTTTTTTTTTTTTTTTTTTV
#11: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCACGACTCCTTTTTTTTTTTTTTTTTTTTTTTTV
#12: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTGATGTCCTTTTTTTTTTTTTTTTTTTTTTTTV
#13: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCATCAGAATTTTTTTTTTTTTTTTTTTTTTTTV
#14: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGTCGTGAATTTTTTTTTTTTTTTTTTTTTTTTV
#15: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCACGACGAATTTTTTTTTTTTTTTTTTTTTTTTV
#16: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTGATGGAATTTTTTTTTTTTTTTTTTTTTTTTV
#17: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTCACACGCTTTTTTTTTTTTTTTTTTTTTTTTV
#18: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCGTGTCGCTTTTTTTTTTTTTTTTTTTTTTTTV
#19: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGACACCGCTTTTTTTTTTTTTTTTTTTTTTTTV
#20: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCATGTGCGCTTTTTTTTTTTTTTTTTTTTTTTTV
#21: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTCACATCATTTTTTTTTTTTTTTTTTTTTTTTV
#22: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCGTGTTCATTTTTTTTTTTTTTTTTTTTTTTTV
#23: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGACACTCATTTTTTTTTTTTTTTTTTTTTTTTV
#24: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCATGTGTCATTTTTTTTTTTTTTTTTTTTTTTTV
#25: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTCACAGAGTTTTTTTTTTTTTTTTTTTTTTTTV
#26: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCGTGTGAGTTTTTTTTTTTTTTTTTTTTTTTTV
#27:CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGACACGAGTTTTTTTTTTTTTTTTTTTTTTTTV
#28: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCATGTGGAGTTTTTTTTTTTTTTTTTTTTTTTTV
#29: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCTAACCGCTTTTTTTTTTTTTTTTTTTTTTTTV
#30: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGCTTGCGCTTTTTTTTTTTTTTTTTTTTTTTTV
#31: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCAGCCACGCTTTTTTTTTTTTTTTTTTTTTTTTV
#32: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTAGGTCGCTTTTTTTTTTTTTTTTTTTTTTTTV
#33: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCTAACTCATTTTTTTTTTTTTTTTTTTTTTTTV
#34: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGCTTGTCATTTTTTTTTTTTTTTTTTTTTTTTV
#35: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCAGCCATCATTTTTTTTTTTTTTTTTTTTTTTTV
#36: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTAGGTTCATTTTTTTTTTTTTTTTTTTTTTTTV
#37: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCCTAACGAGTTTTTTTTTTTTTTTTTTTTTTTTV
#38: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCGCTTGGAGTTTTTTTTTTTTTTTTTTTTTTTTV
#39:CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCAGCCAGAGTTTTTTTTTTTTTTTTTTTTTTTTV
#40: CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATCTAGGTGAGTTTTTTTTTTTTTTTTTTTTTTTTV

### Page 3

Single cell isolation :
Individual cells (so far we've worked with C. elegans blastomeres or trypsinised tissue culture cells) are
transferred with a micro-pipette into a 0.5µl drop of appropriate buffer (egg salts or PBS) placed on the
cap of a 0.5 ml LoBind Eppendorf tube. Location of cell should be marked. Excess liquid is aspirated off,
and tube is frozen in liquid nitrogen. Samples are stored at -80°C.
RNA Amplification:
Prepare primer mix (for each different primer used):
Primer (25ng/μl) 1μl
ERCC Spike-in          Xμl
water Yμl
6μl
Spike-in dilution should be appropriate for sample size - see protocol of ERCC RNA spike in mix. For
single cells we add 1ul of spike-in at 1:500,000 dilution.
Breaking cell open and annealing with primer:
 Add 1.2μl primer mix to marked location of single cell on cap of tube (Keep cell frozen until
adding the primer mix, handle up to 12 cells in parallel).
 Incubate 5 min. at 70 o C (with lid of thermal cycler set to 70 o C).
 Brief spin down.
 Incubate for an additional 5 min. at 70 o C.
 Move immediately to ice.
 Spin at maximal speed for a few seconds to collect as many droplets as possible before next
step, and then return to ice.
RT reaction (Ambion kit)
 Add 0.8μl of the following mix to each reaction:
First Strand buffer 0.2μl
dNTP 0.4μl
RNase Inhibitor 0.1μl
ArrayScript 0.1μl
 Incubate 2hr at 42 o C ( in hybridization oven)
Second strand reaction (Ambion kit):
 Move previous step to ice so it cools below 16 o C.
 Add 8uL of the following mix to each reaction tube:

### Page 4

DDW 6.3μl
Second strand buf. 1μl
dNTP 0.4μl
DNA Pol 0.2μl
RNaseH 0.1μl
Flick and spin samples (at maximal speed for a few seconds).
 Incubate at 16 o C for 2hr (in thermal cycler with unheated or open lid).
cDNA cleanup and speedvac:
 Pool all cells that are to go to same IVT.  Should have ~10μl from each cell.
 Adjust volume to 100μl with nuclease free water.  If more than 10 cells in a pool, just add all
together.
 Add 250μl cDNA binding buffer to each 100ul sample (if total sample volume exceeds 100uL,
adjust cDNA binding buffer volume according to sample volume). Load onto Ambion cDNA
cleanup column. Volume of up to 24 pooled samples can be loaded. If more than 24 samples are
pooled, after spin load remaining volume and spin again.
 Spin 1 min at 10,000g to bind cDNA, discard flow through (repeat if more than 24 samples are
pooled).
 Add 500μl wash buffer, spin as above, discard flow through.
 Spin for an additional minute to dry.
 Transfer column to clean round bottom 2 ml tube.
 Elute by adding 9μl warm water (55 o C), incubating for 2 minutes at room temperature and
spinning 1.5 min at 10,000g.
 Repeat elution.
 Adjust volume to 6.4μl by drying in a speedvac (~8 min).
Stopping point: Samples can be kept at -20 o C
IVT (Ambion kit):
 Prepare the following mix and add 9.6μl per tube.
A 1.6μl
G 1.6μl
C 1.6μl
U 1.6μl
10xT7 buffer 1.6μl
T7 enzyme 1.6μl
 Incubate in a thermal cycler at 37 o C for 13 hrs, with lid at 70 o C.  Set cycler to go to 4 o C at end of
incubation. aRNA (amplified RNA) is stable for at least several hours.

### Page 5

RNA fragmentation and cleanup:
 Mix the following on ice:
aRNA 16μl
Fragmentation buffer 4μl
 Incubate for 3 min. at 94 o C.
 Immediately move to ice and add 2μl fragmentation stop buffer.
 Adjust volume to 30ul by adding 8μl water.
 Add 105μl aRNA binding buffer, followed by 75μl EtOH, immediately mix by pipetting 3-4 times
and load onto spin-column. Bind sample by immediately spinning for 1 min at 10,000g, discard
flow through. (If cleaning more than one reaction, this step should be done for each sample
separately)
 Add 500μl wash buffer (samples can wait at this step until binding of all samples is complete).
 Spin as above, discard flow through.
 Spin for an additional minute to dry.
 Transfer column to clean tube.
 Elute by adding 10μl warm water (55 o C), incubating for 2 minutes at room temperature and
spinning 1.5 min at 10,000 g.
 Repeat elution.
Stopping point: Samples can be kept at -80 o C
Check aRNA amount and quality:
 Load 1μl onto Bioanalyzer RNA pico chip after heating an aliquot of the sample to 70 o for 2 min.
 When starting the IVT with ~0.5ng total RNA, the expected yield is 500-1000 pg/μl. Size
distribution should peak at ~500 bp (See Bioanalyzer plot for example).

### Page 6

Library preparation:
Protocol designed for 5-10ng amplified RNA, although as little as 1-2ng can be used, but then additional
PCR cycles are required. Sample volume should be adjusted to 16μl, either by adding water or drying
down in a speedvac, depending on RNA concentration. IVTs can be pooled at this point if there is no
overlap in barcodes used.
Phosphatase treatment:
To 16μl of fragmented aRNA in a 0.7ml PCR tube add 4μl of the following mix:
 10X phospatase buffer 2μl
 Antarctic phosphatase 1μl
 RNaseOUT 1μl
Incubate in a thermal cycler with the following protocol:
 37°C for 30 minutes
 65°C for 5 minutes
 4°C indefinite hold
PNK treatment:
To the 0.7 ml PCR tube from the previous step add 30μl of the following mix:
 nuclease-free H2O 17μl
 10X phosphatase buffer 5μl
 ATP (10mM, from Illumina kit) 5μl
 RNaseOUT 1μl
 PNK 2μl
Incubate in a thermal cycler at 37°C for 60 minutes then 4°C hold.
Column cleanup of phosphatase and PNK treated aRNA (RNeasy kit):
 Adjust the volume to 100μl (add 50μL) using nuclease free water.
 Add 350μl of RLT buffer and mix well.
 Add 250μl EtOH, mix well by pipetting, and transfer sample to an RNeasy spin column.
 Spin 15 sec. at 8,000 g.
 Transfer column to new collection tube, and add 500μl Buffer RPE.
 Spin 15 sec. at 8,000 g.
 Discard flow-through, and add 500 ml 80% EtOH.
 Spin 2 min. at 8,000 g.
 Transfer column to new collection tube, open lid of column, and spin 5 min at full speed.
 Transfer column to new collection tube, and elute with 14μl nuclease free H2O, spinning at full
speed for 1 minute.
 Dry down the sample using a speedvac to 5μl. (approx. 7 minutes)

### Page 7

Ligate 3' adapter:
Dilute 3' adapter (RA3, from Illumina kit) 5 fold.
To 5μl phosphatase and PNK treated RNA add 1μl of the diluted 3' adaptor.
Incubate at 70°C for 2 minutes and then immediately place the tube on ice to prevent secondary
structure formation.
Add 4μl of the following mix:
 5X HM Ligation Buffer (HML, Illumina kit) 2μL
 RNase Inhibitor (Illumina kit) 1μL
 T4 RNA Ligase 2, truncated 1μL
Incubate the tube on the pre-heated thermal cycler at 28°C for 1 hour (with unheated or open lid).
With the reaction tube remaining on the thermal cycler, add 1μl Stop Solution (STP, Illumina kit) and
gently pipette the entire volume up and down 6-8 times to mix thoroughly. Continue to incubate the
reaction tube on the thermal cycler at 28°C for 15 minutes, and then place the tube on ice.
Add 3μL nuclease free water.
Reverse transcription reaction:
Dilute dNTPs (from Illumina kit) two fold with nuclease free water (prepare at least 1μl per sample)
Combine the following in a PCR tube (the remaining 3' adapter-ligated RNA may be stored at -80°C):
 Adapter-ligated RNA 6μL
 RNA RT Primer (RTP, from Illumina kit)   1μL
Incubate the tube at 70°C for 2 minutes and then immediately place the tube on ice.
Add 5.5μl of the following mix:
 5X First Strand Buffer 2μL
 12.5 mM dNTP mix (diluted dNTP) 0.5μL
 100 mM DTT 1μL
 RNase Inhibitor (Illumina kit) 1μL
 SuperScript II Reverse Transcriptase 1μL
Incubate the tube in the pre-heated thermal cycler at 50°C for 1 hour and then place the tube on ice.

### Page 8

PCR amplification:
To each reverse transcription reaction add 35.5μl of the following mix:
 Ultra Pure Water 8.5μL
 PCR mix (PML, from Illumina kit)) 25μL
 RNA PCR Primer (RP1, from Illumina kit) 2μL
To each reaction add 2μl of a uniquely indexed RNA PCR Primer (RPIX, from Illumina kit)
Amplify the tube in the thermal cycler using the following PCR cycling conditions:
 30 seconds at 98°C
 12 cycles of:
 10 seconds at 98°C
 30 seconds at 60°C
 30 seconds at 72°C
 10 minutes at 72°C
 Hold at 4°C
Can go up to 15 cycles if necessary, or down to 11 if starting with the full 10ng.
Stopping point: samples can be kept at -20 o C.
Bead Cleanup of PCR products - Repeat 1:
 Prewarm beads to room temperature.
 Vortex AMPure XP Beads until well dispersed, then add 50μl to the 50μl PCR reaction. Mix entire
volume up ten times to mix thoroughly.
 Incubate at room temperature for 15 min.
 Place on magnetic stand for at least 5 min, until liquid appears clear.
 Remove and discard 95μl of the supernatant.
 Add 200μl freshly prepared 80% EtOH.
 Incubate at least 30 seconds, then remove and discard supernatant without disturbing beads.
 Add 200μl freshly prepared 80% EtOH
 Incubate at least 30 seconds, then remove and discard supernatant without disturbing beads.
 Air dry beads for 15 min, or until completely dry.
 Resuspend with 32.5μl Resuspension Buffer (from Illumina kit). Pipette entire volume up and
down ten times to mix thoroughly.
 Incubate at room temperature for 2 min.
 Place on magnetic stand for 5 min, until liquid appears clear.
 Transfer 30μl of supernatant to new tube.

### Page 9

Bead Cleanup of PCR products - Repeat 2:
Repeat as above, but adding 39μl beads and eluting in 12.5μl resuspension buffer at the end,
transferring 10μl to a new tube.
Check library amount and quality:
Check concentration of DNA by Qubit, 1μl should be enough to measure using the high sensitivity
reagent; expected concentration is at least ~1ng/μl.
Run 1μl of each sample on Bioanalyzer using a high sensitivity DNA chip to see size distribution.
Expected peak at 300-400bp (See Bioanalyzer plot for example).
Concentration to be loaded for sequencing should be calibrated by the sequencing facility. For us, 5pM
on Hi-Seq v.1 reagents gave good cluster density.