|
| Status |
Public on Aug 01, 2017 |
| Title |
A384T Treg_3 (ChIP-seq) |
| Sample type |
SRA |
| |
|
| Source name |
Treg cells from FoxP3A384T:hCD2 mice
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
tissue: lymph nodes and spleen cells
cell type: CD4(+)hCD2(+) Treg cells
sorted from: FoxP3A384T:hCD2 mice
chip antibody: FoxP3 (affinity purified rabbit polyclonal antibody, generated in house)
|
| Treatment protocol |
none
|
| Growth protocol |
freshly isolated cells
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Magnetically sorted hCD2(+) WT and A384T Treg cells (approx. 1x10e7 cells per reaction, prepared in triplicates) were fixed for 30 min at room temperature with 1% formaldehyde, washed with PBS, and stored at -80 °C. Cells were resuspended in lysis buffers and sonicated for solubilization and shearing of crosslinked DNA. The cell extract was incubated overnight at 4 °C with 4 µg of the anti-FoxP3 antibodies and then with Dynabeads protein G (Dynal). Bound complexes were washed and eluted from the magnetic beads by heating at 65°C with occasional vortexing and crosslinking was reversed by overnight incubation at 65°C. The immunoprecipitated DNA and whole-cell extract input DNA were treated with RNase A and proteinase K, purified using MinElute PCR purification kit (QIAGEN)
ChIP-seq libraries were prepared using NEBNext ChIP-Seq Library Prep Reagent Set for Illumina (NEB) according to the manufacturer's instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
Basecalls performed using Illumina offline basecaller (OLB) version 1.9.4.
DNA sequence reads were aligned to the mouse reference genome (UCSC mm9) using the Bowtie 2 software (Langmead and Salzberg, 2012).
FoxP3 ChIP-seq peaks were called with Model-based Analysis for ChIP-Seq (MACS) (Zhang et al., 2008) (v2.1.0) using input chromatin as control and FDR q-value of <10e-10.
Genome_build: mm9
Supplementary_files_format_and_content: Tabular files in txt format generated by the MACS2 peak-calling program, which contains information about called peaks.
|
| |
|
| Submission date |
Nov 10, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Shohei Hori |
| E-mail(s) |
shohei.hori@riken.jp
|
| Phone |
+81-45-503-7069
|
| Organization name |
RIKEN Center for Integrative Medical Sciences
|
| Lab |
Laboratory for Immune Homeostasis
|
| Street address |
1-7-22 Suehiro-cho, Tsurumi-ku
|
| City |
Yokohama |
| State/province |
Kanagawa |
| ZIP/Postal code |
230-0045 |
| Country |
Japan |
| |
|
| Platform ID |
GPL18480 |
| Series (2) |
| GSE89743 |
Genome-wide maps of FoxP3 binding in FoxP3WT and FoxP3A384T Treg cells |
| GSE89744 |
Analyses of a mutant FoxP3 allele reveal BATF as a critical transcription factor in the differentiation and accumulation of tissue regulatory T cells |
|
| Relations |
| BioSample |
SAMN06008921 |
| SRA |
SRX2341419 |
