|
| Status |
Public on May 14, 2024 |
| Title |
CREB_DMSO_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
primary CD8 T cell
|
| Organism |
Mus musculus |
| Characteristics |
cell type: primary CD8 T cell
genotype: WT
treatment: DMSO
cut-tag antibody: anti-CREB1 (proteintech, 12208-1-AP)
|
| Treatment protocol |
After 24 hours of activation by anti-CD3/CD28, activated CD8+ T cells were treatment with DMSO or Yoda1 (10 M) for another 24h.
|
| Growth protocol |
CD8+ T cells were enriched from the spleens and peripheral lymph nodes of donor mice using a naïve CD8+ T cell isolation kit. Cells were resuspended at 106 per ml in RPMI1640 complete medium with 10% FBS (Gibco), 50 M β-mercaptoethanol, 100 U ml-1 penicillin-streptomycin, non-essential amino acids (Gibco) and sodium pyruvate (Gibco).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
1 × 10^5 CD8+ T cells were collected and washed once with 500 μl wash buffer before they were bound to ConA beads for 10 min at 25 °C.
Cells were incubated with 1 mg of respective targeting antibodies at 4 °C overnight. 0.5 mg secondary antibody was added and incubated for 1 h at 25 °C. Then cells were washed three times with DIG wash buffer and incubated with 0.04 μM pA/G–Tnp for 1 h at 25 °C. Similarly, cells were washed three times with DIG 300 buffer, resuspended in tagmentation buffer and incubated at 37 °C for 1 h. Tagmentation was stopped by adding proteinase K, buffer LB and DNA extract beads. After incubation at 55 °C for 10 min, cells were plated on a magnet and unbound liquid was removed. Beads were gently rinsed twice with 80% ethanol and DNA was eluted with double-distilled water. libraries were barcoded using TruePrep™ Index Kit V2 for lllumina (Vazyme TD202) and amplified for 14 cycles. Final product was cleaned by VAHTS DNA Clean Beads at a 2 × ratio. Libraries were sequenced on a Novaseq 6000 in a 150 bp/150 bp Paired end run. An average of 20 × 106 paired reads was generated per sample.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
CREB_D2
|
| Data processing |
Raw sequencing reads were trimmed and filtered for quality using CutAdapt (v.2.6). Paired-end reads were aligned using Bowtie2 (v.2.3.5.1) (PMID: 22388286) against mm10 genome.
Aligned reads were sorted and non-uniquely mapping reads were marked using Picard (v.2.9.5) (http://broadinstitute.github.io/picard/). PCR duplicates, reads mapped to mitochondria or of low mapping quality were filtered using Samtools (v.1.9) (PMID: 19505943).
To correct for the fact that the Tn5 transposase binds as a dimer and inserts two adapters in the Tn5 tagmentation step, all positive-strand reads were shifted 4 bp downstream and all negative-strand reads were shifted 5 bp upstream to center the reads on the transposon binding event.
We then pooled the shifted reads by sample type and identified peaks using MACS2 (v.2.2.6) (PMID: 18798982) with a threshold of FDR-corrected p < 0.001 using the Benjamini-Hochberg procedure for multiple hypothesis correction. A union peak list for each experiment was created by combining all peaks in all samples; overlapping peaks were merged with minimum overlap > 0.5. The number of reads in each peak was determined with Diffbind (v.3.6.3) (PMID: 22217937).
Differentially expressed peaks were identified after DESeq2 normalization using a FDR cut-off of 0.05. Peaks were annotated using R package ChIPseeker (v.1.18.0) (PMID: 25765347).
Assembly: mm10
Supplementary files format and content: BigWig Track Format of each CUT&Tag sample.
Library strategy: CUT&Tag
|
| |
|
| Submission date |
Feb 28, 2024 |
| Last update date |
May 14, 2024 |
| Contact name |
Dawang Zhou |
| E-mail(s) |
dwzhou@xmu.edu.cn
|
| Organization name |
Xiamen University
|
| Department |
Life Science
|
| Lab |
Dawang Zhou Lab
|
| Street address |
Xiang'an south rd, Xiang'an district
|
| City |
Xiamen |
| State/province |
Fujian |
| ZIP/Postal code |
361102 |
| Country |
China |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE260449 |
Osr2 functions as a mechanical checkpoint to augment CD8+ T cell exhaustion [YODA1_vs_DMSO_CutnTag_CD8] |
|
| Relations |
| BioSample |
SAMN40187146 |
| SRA |
SRX23777809 |
