10x GENOMICS CG000473 Chromium Next GEM Automated Single Cell 5ft cDNA Kit User Guide
- June 3, 2024
- 10x GENOMICS
Table of Contents
SUPPLEMENTAL USER GUIDE Chromium
Next GEM Automated Single Cell 5ʹ cDNA Kit v2
User Guide
FOR USE WITH
Chromium Next GEM Automated Single Cell 5’ cDNA Kit v2, 24 rxns PN-1000425
PAIRS WITH DOCUMENTS
Chromium Next GEM Automated Single Cell 5’ Reagent Kits v2 User Guide
(CG000384)
Chromium Connect Instrument User Guide (CG000180)
Automated Gene Expression Library Construction User Guide (CG000474)
For Chromium Next GEM Automated Single Cell 5′ v2 with Feature Barcode technology for Cell Surface Protein/Immune Receptor Mapping Use the Chromium Automated Single Cell 5′ Feature Barcode Library Construction Kit, 24 rxns PN-1000455. Refer to the Chromium Next GEM Automated Single Cell 5′ Reagent Kits v2 with Feature Barcode technology for Cell Surface Protein & Immune Receptor Mapping User Guide (CG000507).
Document Number
CG000473 Rev B
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Document Revision Summary
| Document Number | CG000473 |
|---|---|
| Title | Chromium Next GEM Automated Single Cell 5′ cDNA Kit v2 Supplemental |
User Guide
Revision| Rev B
Revision Date| Mar-22
Specific Changes| • Updated to include information regarding compatibility
with Chromium Next GEM Automated Single Cell 5′ v2 with Feature Barcode
technology for Cell Surface Protein & Immune Receptor Mapping (pages 1, 5, 8,
9)
• Updated to include chip kit information (page 8)
Specific Changes| • Updated for general minor consistency of language and
terms throughout
Automated Modular Workflow
Chromium Next GEM Automated Single Cell 5′ cDNA Kit v2 Workflow Overview
Chromium Next GEM Automated Single Cell 5′ cDNA Kit v2
Protocol Steps & Timing
Chromium Next GEM Automated Single Cell 5′ cDNAKit v2 Workflow
Overview
This is a supplemental document that provides an overview of the Chromium
Next GEM Automated Single Cell 5′ Gene Expression modular workflow along with
a list of 10x Genomics reagents needed to generate cDNA from single cell
suspension. As part of this automated flexible workflow, cDNA** can be
generated using the Chromium Next GEM Automated Single Cell 5′ cDNA Kit v2
(PN-1000425). The cDNA can be stored at 4°C or -20°C or used directly for
Single Cell 5′ Library Construction using the Automated Library Construction
Kit (PN-1000428) as described in the Automated Gene Expression Library
Construction User Guide (CG000474).
For information on sample preparation, carrier loading and protocol
guidelines, refer to the Chromium Next GEM Automated Single Cell 5′ Reagent
Kits v2 User Guide (CG000384) and the instrument user interface.
For instrument operation, refer to the Chromium Connect Instrument User Guide
(CG000180).
Automated Single Cell 5′ Gene Expression Modular Workflow Overview
| Workflow Step | Kit Name & Documents |
|---|---|
| Use Chromium Next GEM Automated 5′ cDNA kit v2 (PN-1000425) to generate |
cDNA**
See next section for a list of 10x Genomics reagents
| Store cDNA at
4°C for ≤72 h or
-20°C for ≤4 wks
| Use Automated Library Construction Kit
(PN-1000428) to construct Gene Expression libraries
Automated Library Construction User Guide (CG000474)
Chromium Next GEM Automated Single Cell 5′ Reagent Kit v2 User Guide
(CG000384)
Chromium Connect Instrument User Guide (CG000180)
Manual
Automated
* If assessing Cell Surface Protein Expression/Immune Receptor Mapping:
Ensure that the samples are labeled as per Demonstrated Protocols CG000149 or CG000203 To simultaneously generate cDNA from mRNA and DNA from the Feature Barcode labeling the Cell Surface Protein, use Feature cDNA Primers 4 (PN-2000277) included in the Automated 5′ Feature Barcode Primer Kit v2 (PN-1000454). Refer to the Chromium Next GEM Automated Single Cell 5′ Reagent Kits v2 with Feature Barcode technology for Cell Surface Protein & Immune Receptor Mapping User Guide (CG000507) for more information.
Chromium Next GEM Automated Single Cell 5ʹ cDNA Kit v2
All reagent tube strips & tubes are for one time use only. DO NOT reuse.
Chromium Next GEM Automated Single Cell 5′ cDNA Kit v2, 24 rxns
PN-1000425
Reagent volumes and colors are different in each of the module types, not all
module tubes contain reagents.
Chromium Next GEM Automated Single Cell Kit, cDNA Module 1, 24 rxns
PN-1000395 (store at 4°C)
Chromium Next GEM Automated Single Cell Kit, cDNA Module 2, 24 rxns
PN-1000396 (store at -20°C)
Chromium Next GEM Automated Single Cell 5ʹ Kit v2, cDNA Module 3, 24 rxns
PN-1000398 (store at -20°C)
Chromium Next GEM Automated Single Cell 5′ cDNA Kit v2, 24 rxns PN-1000425
Chromium Next GEM Automated Single Cell 5ʹ Gel Bead Kit v2, 24 rxns
PN-1000291 (store at -80°C)
Chromium Next GEM Chip K Automated Single Cell Kit, 48 rxns PN-1000289 (store at ambient temperature)
Chromium Partitioning Oil
PN
Partitioning Oil 6 2000190
Chromium 50% Glycerol
PN
50% Glycerol 6 2000109
Chromium
Next GEM Chip K
Automated Single Cell| # PN
---|---
Next GEM Chip K
Automated Single Cell (gasket attached)| 6 2000371
Chromium Next GEM Chip K Automated Single Cell Kit, 16 rxns PN-1000297 (store at ambient temperature) 10xGenomics.com
Chromium Partitioning Oil
PN
Partitioning Oil 2 2000190
Chromium 50% Glycerol
PN
50% Glycerol 2 2000109
Chromium
Next GEM Chip K Automated Single Cell| # PN
---|---
Next GEM Chip K
Automated Single Cell (gasket attached)| 2 2000371
Use the 10x Genomics reagents listed above for automated cDNA* generation.
After cDNA generation, store cDNA until ready to proceed to Gene Expression
Library Construction using the Automated Library Construction Kit
(PN-1000428). Refer to the Automated Gene Expression Library Construction User
Guide (CG000474) for details.
For Chromium Next GEM Automated Single Cell 5′ v2 with Feature Barcode
technology for Cell Surface Protein/Immune Receptor Mapping, use the Chromium
Automated Single Cell 5′ Feature Barcode Library Construction Kit
(PN-1000455). Refer to the Chromium Next GEM Automated Single Cell 5′ Reagent
Kits v2 with Feature Barcode technology for Cell Surface Protein & Immune
Receptor Mapping User Guide (CG000507) for details.
Protocol Steps & Timing
Steps Timing
Manual
| Cell Preparation (Dependent on Cell Type) | ~1-1.5 h |
|---|---|
| Gather & Load Reagents and Consumables | ~60 min |
Automated Single Cell Gene Expression cDNA
Master Mix Preparation
Chromium Automated Controller Loading
GEM Generation
OPTIONAL
Confirm GEM Generation (Manual, 5 min)
| Post GEM RT-Cleanup – Dynabead | ~4.5 h |
|---|---|
| cDNA Amplification* | Walk-away |
| cDNA Cleanup – SPRIselect | time |
Stop after SPRI clean up, store cDNA at 4°C for 72 hrs or -20°C for 4 wks
cDNA QC & Quantification (Manual, 50 min; best practice)
Automated Single Cell Gene Expression Library
Fragmentation, End Repair & A-tailing
Post Fragmentation, End Repair & A-tailing Double Sided Size Selection
| SPRIselect | ~4 h |
|---|---|
| Adaptor Ligation | Walk-away |
| Post Ligation Cleanup- SPRIselect | time |
Sample Index PCR
Post Sample Index PCR Double Sided Size Selection- SPRIselect
Sample Index PCR
Post Sample Index PCR Double Sided Size Selection- SPRIselect
OPTIONAL
Library Quantification qPCR & Library Pooling
*For Chromium Next GEM Automated Single Cell 5′ v2 with Feature Barcode technology, cDNA from mRNA and DNA from the Feature Barcode will be generated simultaneously. Remove the supernatant from the instrument deck and store at 4°C or at −20°C for up to 5 weeks before proceeding to the next automated step (Feature Barcode DNA Supernatant – SPRIselect Cleanup). Refer to the Chromium Next GEM Automated Single Cell 5′ Reagent Kits v2 with Feature Barcode technology for Cell Surface Protein & Immune Receptor Mapping User Guide (CG000507) for details.
Chromium Next GEM Automated Single Cell 5ʹ
cDNA Kit v2 Supplemental User Guide • Rev B
Documents / Resources
| 10x
GENOMICS CG000473 Chromium Next GEM Automated Single Cell 5ft cDNA
Kit
[pdf] User Guide
CG000473 Chromium Next GEM Automated Single Cell 5ft cDNA Kit, CG000473,
Chromium Next GEM Automated Single Cell 5ft cDNA Kit
---|---