Mar 04, 2025
Single-cell RNA-sequencing
This protocol is a draft, published without a DOI.
- Alexander Misharin1
- 1Northwestern University
Protocol Citation: Alexander Misharin 2025. Single-cell RNA-sequencing. protocols.io https://protocols.io/view/single-cell-rna-sequencing-d4378yrn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 04, 2025
Last Modified: March 04, 2025
Protocol Integer ID: 123743
Keywords: scrna libraries on illumina nextseq, sequencing scrna library, single cell rna, scrna, rna, cell rna, illumina nextseq, sequencing protocol, sequencing
Abstract
Protocol for preparing and sequencing scRNA libraries on Illumina NextSeq 500.
Troubleshooting
Load 10,000 cells from each sample into a separate channel of a
Equipment
Chromium Next GEM Chip A Single Cell Kit
NAME
10X Genomics
BRAND
120236
SKU
for use in the
Equipment
Chromium Controller
NAME
microfuidic based single-cell capture
TYPE
10x Genomics
BRAND
1000171
SKU
LINK
Partition cells into nanoliter scale Gel Beads in emulsions (GEMs) and lyse cells using the
Equipment
Single Cell 3' Chip V3.1 GEM
NAME
10X Genomics
BRAND
1000121
SKU
10x Genomics Single Cell 3′ Chip V3.1 GEM, Library and Gel Bead Kit
Reverse transcribe RNA and amplify using 8 cycles of PCR
Prepare the libraries using
Equipment
Chromium Single Cell 3' Library & Gel Bead Kit
NAME
Library & Gel Bead Kit
TYPE
10X Genomics
BRAND
120267
SKU
Include between 15-20 ng of cDNA in the solution. Amplify using 14 rounds of PCR amplification
Sequence the libraries on
Equipment
NextSeq 500 System
NAME
Sequencer
TYPE
Illumina
BRAND
*********
SKU
LINK
.