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## 10x Genomics Accessories |
| Product | Part Number (Kit) | Part Number (Item) | |
|-------------------------------------------------------------------------------|---------------------------------------------------------------------------|----------------------| |
| 10x Vortex Adapter | 120251 | 330002 | |
| 10x Magnetic Separator B* | 1000709 (Chromium X/iX Series Accessory Kit)/ | 2001212 | |
| Chromium X/iX Chip Holder (also referred to as Chromium X Series Chip Holder) | 1000821 (Chromium X Series Accessory Kit)/ 1000707 (GEM-X Transition Kit) | 3000598 | |
## Third-Party Items |
Successful execution of Chromium Gene Expression Solutions requires thirdparty reagents, kits, and equipment in addition to those provided by 10x Genomics. All third-party reagents and consumables should be obtained prior to starting this library construction workflow. |
Refer to the Chromium GEM-X Single Cell Gene Expression v4 & Immune Profiling v3 - Protocol Planner (CG000748) for a detailed list of the following third-party items: |
- l Additional reagents, kits, and equipment |
- l Recommended pipette tips |
- l Recommended thermal cyclers |
10x Genomics has tested all items listed in the Protocol Planner. These items perform optimally with the assay. Substituting materials may adversely affect assay performance. |
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## Protocol Steps & Timing |
| Steps | Timing | Stop & Store | |
|----------------------------------------------------------------------------------------|-----------|----------------------------| |
| Sample Preparation | variable* | Demonstrated Protocols for | |
| Step 1: GEM Generation and Barcoding (page 29) | | | |
| 1.1 Prepare Master Mix (page 31) | 20 min | | |
| 1.2 Load GEM-X Chip (page 36) | 10 min | | |
| 1.3 Run the Chromium X Series Instrument (page 38) | 6 min | | |
| 1.4 Transfer GEMs (page 39) | 3 min | | |
| 1.5 GEM-RT Incubation (page 40) | 55 min | 4°C ≤72 h/-20°C ≤1 week | |
| Step 2: Post GEM-RT Cleanup & cDNA Amplification (page 41) | | | |
| 2.1 Post GEM-RT Cleanup - Dynabeads (page 43) | 45 min | | |
| 2.2 cDNA Amplification (page 46) | 40 min | 4°C ≤72 h/-20°C ≤1 week | |
| 2.3 cDNA Cleanup - SPRIselect (page 47) | 30 min | 4°C ≤72 h/-20°C ≤4 weeks | |
| 2.4 Post cDNA Amplification QC & Quantification (page 48) | 50 min | | |
| Step 3: 3' Gene Expression Library Construction (page 50) | | | |
| 3.1 GEX Fragmentation, End Repair & A-tailing (page 53) | 50 min | | |
| 3.2 GEX Post Fragmentation, End Repair & A-tailing Double Sided - SPRIselect (page 55) | 30 min | | |
| 3.3 GEX Adaptor Ligation (page 56) | 25 min | | |
| 3.4 GEX Post Ligation Cleanup - SPRIselect (page 57) | 30 min | | |
| 3.5 GEX Sample Index PCR (page 58) | 40 min | 4°C ≤72 h | |
| 3.6 Post Sample Index PCR Double Sided Size Selection - SPRIselect (page 59) | 30 min | 4°C ≤72 h/-20°C long term | |
| 3.7 Post Library Construction QC (page 60) | 50 min | | |
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## Stepwise Objectives |
The Chromium GEM-X Single Cell Gene Expression v4 upgrades short read sequencers to deliver a scalable microfluidic platform for 3' digital gene expression by profiling 500-20,000 individual cells per sample. A pool of ~3,600,000 10x Barcodes are sampled separately to index each cell's transcriptome. It is done by pa... |
This document outlines the protocol for generating Single Cell 3' Gene Expression dual index libraries from single cells. |
## GEM-X Single Cell 3' Gel Beads v4 |
In addition to the poly(dT) primer that enables the production of barcoded, full-length cDNA from poly-adenylated mRNA, the GEM-X Single Cell 3' Gel Beads v4 also include an additional primer sequence (Capture Sequence 1) that enables the capture and priming of Feature Barcode technology compatible targets or analyte... |
Only the poly(dT) primers are used in this protocol for generating Single Cell 3' Gene Expression libraries. |
## Gel Bead Primers |
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