 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 28, 2017 |
| Title |
OTId8-aPDL1_r3 |
| Sample type |
SRA |
| |
|
| Source name |
tumor-infiltrating lymphocytes
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J CD45.1 OT-I
cell type: CD8+ T cells
treatment: anti-PD-L1
|
| Treatment protocol |
Total CD8+ T cells, harvested from spleen and lymph nodes from 6-12-week old OT-I or P14 mice by negative selection were activated in vitro by crosslinked anti-CD3 plus anti-CD28. On day 2, T cells were removed from the antibody-coated plate and re-cultured at 400,000 cells/mL with low dose (10 U/ml) IL-2. Every day the cell concentration was adjusted to 400,000 cells/mL until day 5-6, when cells were adoptively transferred into tumor-bearing mice.
|
| Growth protocol |
When the B16-OVA tumor reached a dimeter of 0.25-0.4 cm, Thy1.1+ P14 and CD45.1+ OT-I cells were collected, washed with PBS, and resuspended at a concentration of 20 million cell/ml in PBS, at a 1:1 P14:OT-I ratio. 2-4.5 million CTL were intravenously injected into the retro-orbital vein in anesthetized mice bearing B16-OVA tumors. In selected experiments, anti-PD-L1 Ab (clone #10F.9G2, BioXCell) or control anti-KLH rat IgG2b (BioXCell) were inoculated intra-peritoneally (200 ug/mouse). Two injections of anti-PD-L1 or control IgG were performed, three and six days after adoptive transfer of OT-I CTL.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell suspensions were prepared from tumors after enzyme digestion. For sorting of adoptively transferred OT-I and P14 cells, CD8+ T cells were positively selected from tumors using the Dynabeads FlowComp Mouse CD8 selection kit (Thermo Fisher). To ensure adequate cell yield, samples from multiple mice belonging to the same group were pooled prior to enrichment. Enriched CD8+ T cells were then stained with anti-CD8a (53-6.7), anti-CD45.1 (A20), anti-Thy1.1 (OX-7), anti-PD-1 (29F.1A12), anti-Tim-3 (RMT3-23) and Fixable Viability Dye eFluor780. Tumor infiltrating lymphocytes were isolated as live CD8+CD45.1+ (OT-I) or CD8+Thy1.1+ (P14) on BD FACSARIA III or FUSION sorters. ATAC-seq was performed according to Buenrostro et al. (Nature Methods 2013) with minor modifications: 30,000 to 50,000 sorted CD8+ T cells cells were lysed to extract nuclei. Nuclei were resuspended in 50 ul 1xTD Buffer containing 2.5 μl transposase (Nextera, Illumina). The transposase reaction was conducted for 30 minutes at 37°C, with mild shaking.
Library amplification and barcoding were performed with the library amplification kit (KAPA Biosystems) using Index Primers, designed according to Buenrostro et al. (Nature Methods 2013) at a final concentration of 500 nM. PCR reaction was conducted for 10-11 cycles. Library purification was performed with the MinElute PCR Purification Kit (Qiagen) and library size distribution was assessed using the Bioanalyzer High Sensitivity DNA Kit (Agilent). ATAC-seq libraries were quantified prior to pooling and sequencing using the real-time library quantification kit (KAPA Biosystems).
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
transposase-digested genomic DNA
|
| Data processing |
Basecalls and demultiplexing was performed in Illumina BaseSpace
Sequencing reads were mapped to the mouse genome (mm10) using Bowtie2 with parameters "--non-deterministic --mm --very-sensitive-local -X 2000
Unmapped reads, reads with MAPQ <30 and reads aligned to chrM, chrY or unlocalized/unplaced scaffolds were removed with samtools and awk
Duplicate reads were removed using picard MarkDuplicates
Reads mapping to regions blacklisted by ENCODE (lifted over to mm10) were removed with bedtools
Reads were shifted by +4 bp (positive strand) or -5 bp (negative strand) so that the read start represents the center of the Tn5 transposon binding event
Each read was trimmed to 15 bp to account for the Tn5 occupancy steric hindrance
BigWig files of coverage for individual replicates were computed with deepTools bamCoverage (--binSize 10 --normalizeUsingRPKM --smoothLength 30)
BigWig files were rescaled using TMM normalization on high-accessibility 300-bp wide windows as calculated by the R/Bioconductor package csaw
Genome_build: mm10
Supplementary_files_format_and_content: BigWig files of coverage, normalized using RPKMs with 10-bp bins
|
| |
|
| Submission date |
Dec 29, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Roberto Spreafico |
| Organization name |
University of Califonia Los Angeles
|
| Department |
Institute for Quantitative and Computational Biosciences
|
| Street address |
UCLA
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE93013 |
Exhaustion-associated regulatory regions in CD8+ tumor-infiltrating T cells (ATAC-seq for in-vivo experiments) |
| GSE93014 |
Exhaustion-associated regulatory regions in CD8+ tumor-infiltrating T cells |
|
| Relations |
| BioSample |
SAMN06190445 |
| SRA |
SRX2450562 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2442537_OTId8-aPDL1_r3.bw |
245.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
