|
| Status |
Public on Oct 10, 2022 |
| Title |
Non-responder-Spleen-4-GEX |
| Sample type |
SRA |
| |
|
| Source name |
Spleen
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Spleen
cell type: CD8 T cells
strain: C57BL/6
age: 6-8 weeks
treatment: anti-PD-L1
tumor state: Non-responder
|
| Treatment protocol |
A223 tumors were injected into C57BL/6J mice. When tumor size reached ~250-350mm3, tumor-bearing mice were randomized into two groups: control vs. anti-PD-L1, treated with PBS or anti-PD-L1 monoclonal antibody (mAb), respectively. Tumors and spleens were collected from responding and non-responding mice for CD8 T cell isolation followed by single-cell sequencing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tumors and spleens were harvested from responders (n=4) and non-responders (n=4) and single-cell suspensions were prepared. Single-cell suspensions of tumor and spleen from the first cohort (Responder-1, Non-responder-1) were flow sorted for alive CD8 T cells. Single-cell suspensions from the second, third and fourth cohort (Responder-2-4 and Non-responder-2-4) were subjected to EasySep™ Mouse CD8a Positive Selection Kit II (StemCell Technologies, Catalog#18953) according to manufacturer’s instructions to purify CD8 T cells. Sorted or purified samples were submitted for single cell capture and library preparation.
Cells were loaded into 10× Genomics Chromium Next GEM Chip K (Catalog#1000286) for the 5’ captures. Single-cell gene expression libraries were prepared using Chromium Next GEM Single Cell 5' Kit v2, (Catalogue#1000265) according to the manufacturer’s instructions. 5' libraries were split in half, one for RNA-seq and another half was enriched for TCR sequencing using the Chromium Single Cell Mouse TCR Amplification Kit (Catalogue#1000254).
Samples were sequenced on the Illumina NovaSeq 6000 platform for an estimated read depth of 50,000 reads per cell (5' expression), or 5,000 reads per cell (TCR VDJ).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
10x Genomics
polyA RNA 5' capture GEX
|
| Data processing |
RNA-seq reads were mapped to mm10 (mouse reference genome) using 10× Genomics CellRanger (version 4.0.0) count pipeline and VDJ sequencing reads were mapped to the GRCm38 reference dataset using CellRanger VDJ (version 4.0.0).
Assembly: mm10 (mouse reference genome), GRCm38 reference.
Supplementary files format and content: 10× Genomics CellRanger output files.
Supplementary files format and content: tar archives for GEX Samples include h5 and barcodes.tsv/features.tsv/matrix.mtx files.
Supplementary files format and content: tar archives for VDJ Samples include clonotypes.csv, contig, and vloupe files.
|
| |
|
| Submission date |
Sep 28, 2022 |
| Last update date |
Oct 10, 2022 |
| Contact name |
Jing Hong Wang |
| E-mail(s) |
jhw51@pitt.edu, wangj28@upmc.edu
|
| Phone |
412-864-7728
|
| Organization name |
University of Pittsburgh
|
| Department |
Department of Medicine
|
| Lab |
Jing Hong Wang Lab
|
| Street address |
5117 Centre Avenue, Suite 2.35
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15213-1862 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE214348 |
Divergent outcomes of anti-PD-L1 treatment coupled with host-intrinsic differences in TCR repertoire and distinct T cell activation states in responding versus non-responding tumors |
|
| Relations |
| BioSample |
SAMN31063948 |
| SRA |
SRX17734674 |
