|
| Status |
Public on Feb 13, 2015 |
| Title |
RNAPII-Ser5(P)_ChIPSeq_Treatment |
| Sample type |
SRA |
| |
|
| Source name |
HEK 293T cells_IP_treatment
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK 293T
cell type: non-malignant cell line
treatment: cells treated for three days with 1.0 µM of 5-aza-CdR
chip antibody: RNAPII-Ser5(P) (Active Motif, 61085)
|
| Treatment protocol |
HEK 293T cells were treated for three days with 1.0 µM of 5-aza-CdR. Cells with no drug were grown as controls. Tissue culture medium was changed every day for both control and treated cells, to maintain the drug stability during treatment. After collection, treated cells were remained in culture for a further 30 days.
|
| Growth protocol |
HEK 293T cells were grown in triplicates, in DMEM supplemented with 10% fetal bovine serum and 2 mM Penicillin-Streptomycin at 37°C in 5% CO2. Cells were passaged only when reaching 80-90% confluence, for a total period of one month.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
3 million cells crosslinked in 1% formaldehyde were lysed with Farnham and RIPA cold lysis buffers. Following collection, the crude nuclear preparation was processed in a Bioruptor at the high setting for a total of 15 minutes, in cycles of 30 seconds on/30 seconds off. The chromatin was collected by centrifugation and incubated with 5 µg of RNAPII-Ser5(P) antibody overnight
The immunoprecipitated samples were end-filled using a combination of T4 DNA polymerase and T4 polynucleotide kinase , 3’ -terminal-A extended with Klenow 5'-3' exo minus, and ligated to pre-annealed TruSeq indexed Illumina adapters. Libraries were amplified using Illumina primers and gel extracted for size selection and primer-dimer removal. Before sequencing, libraries were tested using the BioAnalyzer to test quality in terms of size and primer-dimer depletion.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
ChIP-seq reads were aligned by the WASP Pipeline version 3.1.5
MACS version 1.4.2 was used for peak finder and bowtie version 0.12.7 as aligner
Genome_build: hg19
Supplementary_files_format_and_content: txt files containing information on called peaks (IP vs INPUT)
|
| |
|
| Submission date |
Oct 24, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Maria-Paz Ramos |
| Organization name |
Albert Einstein College of Medicine
|
| Department |
Genetics
|
| Lab |
John Greally
|
| Street address |
1301 Morris Park Ave, Price 314
|
| City |
Bronx |
| State/province |
New York |
| ZIP/Postal code |
10461 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE62590 |
DNA demethylation by 5-aza-2'-deoxycytidine is imprinted targeted to euchromatin and has limited transcriptional consequences |
| GSE62695 |
Genome-wide maps of RNAPII Ser5(P) in HEK 293T cells after treatment with 5-aza-CdR |
|
| Relations |
| BioSample |
SAMN03142839 |
| SRA |
SRX742138 |
