|
| Status |
Public on Mar 02, 2013 |
| Title |
RNAPII_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
HCT116 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: colorectal carcinoma
passages: 15-18
cell line: HCT116 cells
chip antibody: RNAPII (Santa Cruz, N-20: sc-899, Lot E1711)
|
| Treatment protocol |
For TAF3, RNAPII, and H3K4me3 ChIP-seq, cells in monolayer were fixed with 1% Formaldehyde for 10 min at room temperature and quenched by adding glycine to a final concentration of 125 mM (Sigma). 10-50 million cells were used per IP.
|
| Growth protocol |
HCT116 cells were cultured on uncoated tissue-culture plates in Dulbecco’s modified Eagle medium (DMEM, Gibco).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated and precleared chromatin was immunoprecipitated with antibody.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~200 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced for 36 bp on the Genome Analyzer IIx following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Basecalls performed using Illumina RTA 1.8.70.0
ChIP-seq reads were aligned to the hg19 genome assembly using Illumina Casava 1.8.2
Data were filtered using the following specifications: only unique alignments were used.
peaks were called using MACS version 1.4.2 with the default setting.
Genome_build: hg19
Supplementary_files_format_and_content: BedGraph files were generated using MACS version 1.4.2 and then converted to bigWig format using the bedGraphToBigWig program. Peaks are filtered by fold_enrichment > 5 and FDR < 0.05.
|
| |
|
| Submission date |
Jan 16, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Xiaolin Wu |
| E-mail(s) |
forestwu@mail.nih.gov
|
| Phone |
301-846-7677
|
| Organization name |
Frederick National Laboratory for Cancer Research/SAIC-Frederick Inc
|
| Department |
Cancer Research Technology Program
|
| Lab |
Genomics Laboratory
|
| Street address |
8560 Progress Drive, C3001
|
| City |
Frederick |
| State/province |
MD |
| ZIP/Postal code |
21701 |
| Country |
USA |
| |
|
| Platform ID |
GPL10999 |
| Series (2) |
| GSE43539 |
H3K4me3 Interactions with TAF3 Regulate Preinitiation Complex Assembly and Selective Gene Activation [ChIP-Seq] |
| GSE43542 |
H3K4me3 Interactions with TAF3 Regulate Preinitiation Complex Assembly and Selective Gene Activation |
|
| Relations |
| SRA |
SRX217740 |
| BioSample |
SAMN01888186 |
