|
| Status |
Public on Nov 14, 2012 |
| Title |
PolII4H8_untreated_Tcell_ChIPseq_pooled5donor |
| Sample type |
SRA |
| |
|
| Source name |
CD4+ T cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: CD4+ T cells
chip antibody: PolII4H8 [Abcam, Cat no. ab5408, Lot no. 722997]
treatment: untreated
|
| Treatment protocol |
Isolated human CD4+ T cells were pooled from five different donors and used for side-by-side experiments of BRD4, Pol II Ser2, Pol II Ser5 and total Pol II (4H8) treated with or without 500nM JQ1 for 24hr.
Freshly isolated human CD4+ T-cells treated with or without 500nM JQ1 for 24hr
|
| Growth protocol |
CD4+ T-cells were purified from normal human peripheral blood using the human CD4+ T-cell Isolation Kit II (Miltenyl Biotech).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP DNA ends were repaired using T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase), and T4 polynucleotide kinase (PNK). The 3' to 5' exonuclease activity of these enzymes removes 3' overhangs and the polymerase activity fills in the 5' overhangs. This is followed by treatment with Klenow fragment (3' to 5' exo minus) to generate an overhanging 3 adenine base for adaptor ligation to adapters which have a single T base overhang at their 3' end. Size Selection of 300bp fragment were done using the Invitrogen EGel system, followed by enrichment with 15 PCR cycles. The amplified DNA was purified by Ampure beads before validation on the Agilent Bionanlyser.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Alignment (bed files): Sequence reads were obtained and mapped to the human (hg18) genomes using Bowtie using default parameters and m 1, k 2, best and strata. All reads mapping with two or fewer mismatches were retained. For ChIP-seq, only unique reads were retained (for reads with duplicates,one read was retained).
Peaks (wig files): Peak detection was performed with FindPeaks4 (Fejes et al., 2008) with an extension of sequence tags to 200bp and a false discovery rate of 0.0000001 using the landerwaterman algorithm.
Normalization (wig normalized files ): Wig files of peaks detected were normalized for comparison between ChIP-seq libraries before and after JQ1 treatment. Tag counts in each position were normalized by a multiplication factor of 10/(Number of million mapped tags in the particular library to 2 decimal places)
Genome Build:
CHT026_CD4TCell_PolIISer5_rmdup.bed: hg18
CHT026_CD4TCell_PolIISer5.wig.gz: hg18
CHT026_CD4TCell_PolIISer5_normalized_40.19.wig.gz: hg18
|
| |
|
| Submission date |
Oct 19, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Keh-chuang Chin |
| E-mail(s) |
[email protected]
|
| Organization name |
Singapore Immunology Network
|
| Department |
Laboratory of Gene Regulation
|
| Street address |
Biopolis, 8A Biomedical Grove, #03-06 Immunos
|
| City |
Singapore |
| ZIP/Postal code |
138648 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE33281 |
Bromodomain-Containing-Protein 4 (BRD4) Regulates RNA Polymerase II Serine 2 Phosphorylation in Human CD4+ T Cells |
|
| Relations |
| SRA |
SRX198035 |
| BioSample |
SAMN01773254 |
