|
| Status |
Public on Mar 30, 2022 |
| Title |
LRF_CHIP2 |
| Sample type |
SRA |
| |
|
| Source name |
CD4+ T cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6Ncr
genotype: BirA
transduction: pMRX-LRFbio-tag-IRES-Thy1.1
cell population: Thy1.1+CD4+
immunoprecipitation: Streptavidin
|
| Treatment protocol |
Live cells were sorted, washed in PBS and fixed in 1% formaldehyde-containing PBS for 5 min at 37 degrees. Following quenching with 0.125M glycine, cells pellets were snap-frozen on dry ice and stored at -80 degrees. Frozen fixed cells were then lysed in 1% SDS RIPA buffer (20 mM Tris-HCl pH 7.6, 2 mM EDTA, 150 mM NaCl, 0.1% sodium deoxycholate, 1% TritonX100) for 30 minutes at 4°C, spun down and sonicated in 0.1% SDS RIPA buffer using a Qsonica Q800R sonicator (30s on, 59s off, 85% amplitude) to obtain a sheared chromatin with an average size of 200bp. Sheared chromatin was pre-cleared with protein-A magnetic beads (Invitrogen 10001D) followed by immunoprecipitation with M280 Streptavidin beads (Invitrogen 11205D). After washing, immunoprecipitated chromatin was reverse-crosslinked by overnight incubation at 70°C in 50nM 50mM Tris-HCl pH 8., 10mM EDTA , 1% SDS buffer
|
| Growth protocol |
Splenic CD4+ T cells from Rosa26BirA or Rosa26BirA animals were enriched using Dynabeads Untouched Mouse CD4 cells kit (Invitrogen) and stimulated with anti-CD3 (1mg/ml), anti-CD28 (3mg/ml) and IL-12 (10ng/ml) . One day after activation, cells were transduced with pMRX-LRFbio-tag-IRES-Thy1.1 retrovirus, or with a control retrovirus expressing Thy1.1 only. Cells were cultured for two additional days with anti-CD3, anti-CD28 and IL-12 and then with IL-2 (100 ng/ml) for another day.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was treated with proteinase K and RNase (both at 0.2mg/ml) before purification using the QIAquick PCR purification kit (Qiagen) Accel-NGS 2S DNA reagent (Swift) Libraries were sequenced with paired-end reads of 76bp on a NextSeq sequencer (Illumina)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Illumina RTA 2.4.11 software used for basecalling.
Samples demultiplexed using Bcl2fastq 2.17 software
Sequenced reads were trimmed for adaptor sequence and low-quality sequence (phred < 32) on the Partek Flow server
Filtered reads were mapped to mm10 whole genome using bowtie v1.0.0 with defauft parameters on the Partek Flow serve
Peaks were called using MACS2 callpeak
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files were generated with the bamCoverage function of the deeptools package
|
| |
|
| Submission date |
May 06, 2020 |
| Last update date |
Mar 30, 2022 |
| Contact name |
Jia Nie |
| E-mail(s) |
jia.nie@nih.gov
|
| Organization name |
National Cancer Institute
|
| Department |
Center for Cancer Research
|
| Lab |
Laboratory of Immune cell Biology
|
| Street address |
Building 37, Room 3016
|
| City |
Bethesda |
| State/province |
Maryland |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE149993 |
LRF genome-wide occupancy in CD4+ T cells |
|
| Relations |
| BioSample |
SAMN14844000 |
| SRA |
SRX8272868 |
