|
| Status |
Public on Apr 24, 2021 |
| Title |
CX6A_1: Single cell 5' gene expression of fresh cevix biopsy/whole cell lysate/subject 6 |
| Sample type |
SRA |
| |
|
| Source name |
Fresh human cervix biopsy
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: single cell suspension from fresh cervix biopsy with one hour plating
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Human ectocervix biopsies were processed into single cell suspension for 10x singel cell immune profiling (5' gene expression + TCR), usually within the four hours after biopsies were collected from the UW virus research clinics (VRC). The process of cervix biopsies were a combination of collagenase, DNase and mechanical agitation (details in the associated manuscript).
Cell suspensions were loaded according to 10x Genomics Manufacturer’s instructions for Chip A to generate Gel Bead-In-Emulsion (GEMS, 10x genomics) using barcoded gel beads. RNA containing GEMS underwent barcoded cDNA synthesis. Subsequent library construction followed 10x Genomics 5’ Single Cell Immune Profiling Manufacturer’s Instructions. Libraries were constructed using the Chromium Next GEM Single Cell 5’ Library & Gel Beads Kit v1.1 with additional Chromium Single Cell V(D)J Enrichment Kit, Human T Cells kit for TCR Libraries.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
One hour plating to remove fibroblast cells before 10x single cell gel bead emulsion
|
| Data processing |
The sequencing of 10x libraries for 5’ GEX and TCR-vdj for the ten cervix biopsies from eight individuals and cell ranger analysis were performed at the shared resource at Fred Hutch. Cell ranger 3.0.2 with human genome GRCh38 for gene expression and GRCh38-alts-ensembl for TCR-vdj were used in the initial steps including quality control, alignment to human genomes and counting of aligned sequence reads for individual genes.
We manually examined summary HTML files for the ten gene expression libraries and the ten TCR-vdj libraries generated from ten cervix samples. All the libraries have valid barcodes (> 85%), fraction reads in cells (> 80%) and reads mapped to genome (> 50%).
The MTX files from the output of cell ranger were imported into Monocle3, Seurat (version 3.1.2) or Scanpy (version 1.4.4) for further analysis. In all the three methods, we filtered out cells with less than 200 genes detected and genes that are only detected in less than 3 cells. We further filtered out cells with percentages of mitochondria genes more than 15%. pyVDJ (https://github.com/veghp/pyVDJ) and Scirpy is used to link TCR-vdj to gene expression in Scanpy.
|
| |
|
| Submission date |
Apr 23, 2021 |
| Last update date |
Apr 25, 2021 |
| Contact name |
Tao Peng |
| E-mail(s) |
tpeng@u.washington.edu
|
| Phone |
206-667-5861
|
| Organization name |
University of Washington
|
| Department |
Medicine
|
| Street address |
1100 Fairview Ave N/FHCRC/E5-110
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE173231 |
Single cell immune profiling of human cervix biopsies |
|
| Relations |
| BioSample |
SAMN18857395 |
| SRA |
SRX10667309 |
