Single-cell sequencing and analysis
Daniele Neri, Lori Zeltser
Published: 2022-07-07 DOI: 10.17504/protocols.io.bp2l61drdvqe/v1
Abstract
This protocol describes single-cell sequencing and analysis.
Steps
Sequencing
1.
We performed plate-based single-cell sequencing as described in Snyder et al., 2019 (https://doi.org/10.1126/sciimmunol.aav5581)
2.
Pooled 3’-end sequencing libraries were sequenced on an Illumina NextSeq 500/550 platform
Alignment and count matrix generation
3.
The reads were aligned to mouse genome, GRCm38, gencode version M13 (Ensembl 88) using STAR v2.5.3a
4.
The aligned reads were assigned to genes using featureCounts v1.5.3.
5.
bam files were sorted and indexed using Samtools v1.4.1, duplicated reads were removed and a UMI count matrix was generated using umi tools
Analysis
6.
We performed analysis of the count matrices using Seurat v2.3.4 in R v3.4.3 (R Core Team, 2013).
