|
| Status |
Public on Dec 20, 2019 |
| Title |
iTreg Foxp3 ChIP |
| Sample type |
SRA |
| |
|
| Source name |
iTreg
|
| Organism |
Mus musculus |
| Characteristics |
cell type: iTreg
strain: Balb/c
chip antibody: anti-Foxp3 (Santa Cruz S-31738 and eBioscience FJK-16)
|
| Treatment protocol |
Cells were activated for 72 hours by plate-bound anti-CD3 and anti-CD28 (both 10 ug/ml; clones 145-2C11 and 37.51, respectively; Bio X Cell). iTreg cells were generated by culturing in recombinant human TGF-β1 (33 ng/ml) and IL-2 (20 ng/ml; R & D Systems) for 7 days. Th1 cells were polarized with 20 ng/ml IL-2 (Biolegend) and polarized with 20 ng/ml IL-12 (eBioscience) and 10 μg/ml anti-IL-4 (BioXCell).
|
| Growth protocol |
CD4+ T cells from spleens and lymph nodes of 4- to 10-wk-old Balb/c mice were purified by CD4 positive selection (Miltenyi Biotec) followed by sorting of naive CD4+CD25-CD62LhighCD44low cells using a FACSAria II (BD Biosciences).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked by the addition of one-tenth volume of fresh 11% formaldehyde solution for 20 minutes at room temperature before the reaction was quenched by addition of glycine. Cells were rinsed twice with 1xPBS and flash frozen in liquid nitrogen. Cells were lysed with non-ionic detergent, nuclei washed and then lysed with ionic detergent. Cells were sonicated on ice to solubilize and shear crosslinked DNA (24W for 10 x 30 second pulses using a Misonix Sonicator 3000). The resulting whole cell extract was cleared by centrifugation and then incubated overnight at 4°C with 100 µl of Dynal Protein G magnetic beads that had been pre-incubated with 5 µg each of 2 Foxp3 antibodies, Santa Cruz S-31738 and eBioscience FJK-16. Beads were washed 6 times with RIPA buffer and 1 time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C with occasional vortexing and crosslinks then reversed in IP and input DNA by overnight incubation at 65°C. IP and input DNA were then purified by treatment with RNase A, proteinase K and phenol:chloroform extraction followed by ethanol precipitation.
Libraries were constructed from ChIP and input DNA by standard Illumina protocols, except that DNA in the range 150-350bp was gel-purified after PCR-amplification. The libraries were quantified using a Qubit and Agilent bioanalyzer, pooled and subjected to 35 or 50 bp single-end read sequencing with an Illumina GAIIx or HiSeq 2000 sequencer
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Basecalls performed using CASAVA
Reads were filtered to remove adapters using fastq-mcf and for quality using seqkt
Reads were then aligned to the mouse genome (mm9) with Bowtie2 (default settings)
Regions of significant enrichment (10^-7) were identified using MACS version 1.4 (Zhang et al., 2008) using input as background, with the setting –keep-dup=1
Genome_build: mm9
Supplementary_files_format_and_content: bed files containing peaks derived from analyses
|
| |
|
| Submission date |
Aug 22, 2015 |
| Last update date |
Dec 21, 2019 |
| Contact name |
Richard Jenner |
| E-mail(s) |
r.jenner@ucl.ac.uk
|
| Organization name |
UCL Cancer Institute
|
| Department |
Cancer Biology
|
| Lab |
Regulatory Genomics
|
| Street address |
72 Huntley Street
|
| City |
London |
| ZIP/Postal code |
WC1E 6BT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE72279 |
microRNA-142–mediated repression of phosphodiesterase 3B critically regulates peripheral immune tolerance |
|
| Relations |
| BioSample |
SAMN04008882 |
| SRA |
SRX1162501 |
