 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 22, 2020 |
| Title |
CASTAT5 spleen CD4+ T cells ATAC-seq 1 |
| Sample type |
SRA |
| |
|
| Source name |
FACS_sorted CD4 T cells
|
| Organism |
Mus musculus |
| Characteristics |
cell surface marker: CD4+Thy1.1+
genotype/variation: Stat5a (H229R & S711F) mutant
strain: Balb/c
|
| Treatment protocol |
A20HA Tumor cells were subcutaneously injected to the right flank of mice. When tumor sizes reached the desired sizes, mice were randomly assigned into groups to receive the specified treatments. Cyclophosphamide (CTX) was i.p. injected to mice at 150 mg/kg one day before T cell transfer. For adoptive CD4 T-cell transfer, in vitro cultured T cells were harvested and injected into each recipient via tail vein.
|
| Growth protocol |
CD4 T cells were purified from 6.5-TCR Tg mouse splenocytes and stimulated with mouse CD3/CD28 activation Dynabeads in the presence of rhIL2 overnight. Cells were harvested and transduced twice in two consecutive days with retroviral supernatant. Cells were harvested 24 hours after the second transduction.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The ATACseq libraries were prepared based on the Omni-ATACseq protocols with some minor modification (Corces MR, Nature Methods volume 14, pages959–962(2017)). Briefly, 60,000 FACS-sorted CD4+ T cells were suspended in 60 ml ATAC- Resuspension Buffer (RSB, 10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin), incubated on ice for 3 minutes, and then washed with 1ml RSB buffer without NP40 and Digitonin. The resulted nuclei were pelleted at 500 RCF for 10 minutes at 4°C in a fixed angle centrifuge and resuspended in 30ml of Tagmentation DNA (TD) buffer. 5ml of the nuclei was removed, mixed with Trypan Blue solution at (1:1) ratio and examined under microscope to confirm a successful nuclei preparation. The remaining 25ml of nuclei was incubated with 2.5ul of Nextera Tn5 transposase (Illumina) in a total volume of 50ml 1× TD buffer containing 0.01% Digitonin and 0.1% Tween-20. The transposition reaction was performed at 37 °C for 50 minutes in a PCR machine. The transposition mixture was purified using a Zymo DNA Clean and Concentrator kit. The ATAC libraries were amplified for 11-13 cycles using NEBNext 2X MasterMix and Nextera Index primers. The amplified libraries were size-selected using AMPure breads (0.5x) to remove the large fragment of DNA and then purified using Zymo DNA Clean and Concentrator kit. Six ATAC libraries were pooled and sequenced on a NextSeq500 instrument in a pair-end 75 cycle run. 60-100 million read pairs were obtained for each sample.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
CASTAT5_SP_rep1
|
| Data processing |
The ATACseq analysis was performed on Galaxy server. Adaptor and quality trimming were performed using Trim Glore! Modules (Galaxy v0.4.3.1). Trimmed reads were mapped to mouse genome mm10 using Bowtie2 (Galaxy v2.3.4.3) using default settings. Reads mapped to the mitochondria genome were removed using Filter SAM or BAM function under the samtools module (Galaxy v1.8), and PCR duplicates were removed using MarkDuplicates from Picard tool v2.20.2. The mapped reads overlapping with ENCODE blacklist regions (version 2) were filtered before calling peaks.
ATAC-seq peak calling was performed on Galaxy server using MACS2 v2.1.2 with following command “-q 0.05 --nomodel --shift --100 --extsize 200 -B --SPMR --keep-dup all --call -summits”.
The Bed file contains common peaks among three biological replicates were generated using bedTools Multiple Intersect function for CASTAT5 and control samples, respectively. The two bed files were combined using bedtools MergeBED function (Galaxy v1.2.0) to generate a single bed file that contains all peaks identified in both CASTA5 and control samples. Finally, the number of paired fragments overlaps with the intervals identified in the merged peak file was counted using bedtools MultiCovBed function (Galaxy v2.27.1) for each of six ATAC-seq samples, respectively.
DEseq2 package v1.24.0 was used to call differential peaks and ChIPseeker v1.20.0. was used annotate the differential peaks.
DiffBind (Galaxy v. 2.6.6.4) was used to identify differential peaks with fixed window length of 400bp (summit ± 200bp), which were used for motif analysis by MEME-ChIP v5.05 and HOMER v4.9.
Genome_build: mm10
Supplementary_files_format_and_content: BigWig files were generated by deepTools bamCoverage function (Galaxy v3.1.2.0.0) and visualized in Integrated genome viewer (IGV) v2.5.2. Normalization/scaling methods: RPGC (1x sequencing depth ) : number of reads per bin /(total number of mapped reads * fragment length / effective genome size). Bin size was set to 10bp.
|
| |
|
| Submission date |
Jun 10, 2020 |
| Last update date |
Oct 22, 2020 |
| Contact name |
Huidong Shi |
| E-mail(s) |
hshi@augusta.edu
|
| Phone |
706-721-6000
|
| Organization name |
Augusta University
|
| Department |
Georgia Cancer Center
|
| Lab |
2125 K
|
| Street address |
1120 15th Street, CN2138
|
| City |
Augusta |
| State/province |
GA |
| ZIP/Postal code |
30912 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE152240 |
Persistent STAT5 activation reprograms the epigenetic landscape in CD4+ T cells to drive polyfunctionality and antitumor immunity [ATAC-Seq] |
| GSE152242 |
Persistent STAT5 activation reprograms the epigenetic landscape in CD4+ T cells to drive polyfunctionality and antitumor immunity |
|
| Relations |
| BioSample |
SAMN15203608 |
| SRA |
SRX8525391 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4609811_239_S4_trim_filter_offset.bigwig |
197.5 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
