|
| Status |
Public on Jan 24, 2017 |
| Title |
Homo sapiens Donor 2 Th1 T-bet |
| Sample type |
SRA |
| |
|
| Source name |
In vitro differentiated Th1 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Th1
antibody: anti-T-bet SY4530FB
treatment/agent: anti-CD3/CD28
|
| Treatment protocol |
Human T cells were formaldehyde crosslinked on day 13 after restimulation witn anti-CD3/CD28.
|
| Growth protocol |
Bulk human CD4+ T-cells were isolated by positive magnetic selection (Miltenyi Biotec). Cells were activated for 72 hours by plate bound anti-CD3 and anti-CD28 antibodies (2 µg/ml, BD Pharmingen) and were then cultured for 10 days with rhIL-2 (Biolegend, 10ng/ml). Conditions for T cell polarization were: rhIL-12 (10 ng/ml, Biolegend) and anti-IL-4 (10 µg/ml, R&D) for Th1.
|
| 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 20 µl of anti-T-bet serum. 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 150 bp single-end read sequencing with an Illumina NextSeq sequencer.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Basecalls performed using CASAVA
Polony identification, base calling and QC statistics were performed using GOAT and Bustard module
Reads (in fastq files) were filtered to remove adapters using fastq-mcf and for quality using seqkt and aligned to the human (hg19) with Bowtie2 (default settings). Bigwig files for visualization in the UCSC genome browser were generated using a custom pipeline; duplicate reads were first removed, coverage calculated with genomeCoverageBed and tag density calculated in 10bp windows. unionBedGraphs was then used to subtract input signals and bigwig files generated using bedGraphToBigWig. Regions of significant enrichment were identified using MACS version 1.4 (Zhang et al., 2008) using input or total histone H3 as background, with the setting --keep-dup=1. A p-value threshold of 10-7 was used, unless stated otherwise.
Genome_build: hg19
Supplementary_files_format_and_content: peak
|
| |
|
| Submission date |
May 25, 2016 |
| Last update date |
May 15, 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 |
GPL18573 |
| Series (1) |
| GSE81881 |
Genetic variants alter T-bet binding and gene expression in mucosal inflammatory disease |
|
| Relations |
| BioSample |
SAMN05176182 |
| SRA |
SRX1799593 |
