|
| Status |
Public on Sep 25, 2015 |
| Title |
SET2_CDK8_untreated_14 |
| Sample type |
SRA |
| |
|
| Source name |
SET2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SET2
cell type: essential thrombocythemia
treatment protocol: untreated
chip epitope: CDK8
chromatin prep#: 14
ca sensitivity: sensitive
|
| Growth protocol |
SET2 cells were grown in RPMI-1640 supplemented with 20% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody.
Libraries for Illumina sequencing were prepared using the Illumina TruSeq ChIP Sample Preparation kit with the following exceptions. After end-repair and A-tailing, ChIP DNA or whole cell extract DNA was ligated to Illumina RNA adaptors with unique indices. Alternatively, libraries were prepared using the KAPA Hyper Prep Kit for Illumina and ligated to unique Bioo Scientific NEXTflex barcode adaptors. Following ligation, libraries were amplified with 16-18 cycles of PCR and were then size-selected using a 2% gel cassette in the Pippin Prep System from Sage Science. For histone modifications and RNA pol II, DNA fragments of size 200-500bp were captured. For CDK8 and MED1, DNA fragments of size 200-450bp were captured. Libraries were quantified by qPCR utilizing the KAPA Biosystems Illumina Library Quantification kit.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
matched input: SET2_input_2_IN5022
|
| Data processing |
Basecalls performed using CASAVA version 1.4
FastQC v.0.10.1 was used to check read quality metrics. If necessary, trim_galore v.0.3.3 was used with default settings to remove adapters, low-quality bases and trimmed reads < 20nt long after trimming.
ChIP-seq reads were aligned to the hg19 genome assembly using bowtie v.1.0.0 with flags -n 1 -m 1 --best --strata
Duplicate reads were removed with MarkDuplicates (picard tools v.1.79(1282))
In cases where peaks were used without subsequent ROSE enhancer analysis, SPP was used both for cross-correlation analysis and for peak finding using default parameters or FDR < 0.05. MACS2 was used either with default parameters or using --shiftsize to set the strand shift to match the SPP peak cross-correlation size. Peaks identified by both peak finders were retained. Regions on chrM or overlapping repeat elements (RepeatMasker bed file downloaded from UCSC 16 Nov. 2012) by > 70% were routinely excluded.
In cases where ROSE was used to identify enhancers and super-enhancers, MACS v.1.4 was run with the suggested parameters, namely -p 1e-9 --keep-dup=auto. MACS v1.4 peak bed files were converted to GFF format and used for ROSE with suggested parameters : -s 12500 -t 2000.
Genome_build: hg19
|
| |
|
| Submission date |
Jan 21, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Matthew D. Shair |
| Organization name |
Harvard University
|
| Department |
Chemistry and Chemical Biology
|
| Lab |
Shair
|
| Street address |
12 Oxford Street
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE65138 |
Effect of cortistatin A (CA) on enhancer occupancy in CA-sensitive and -insensitive human cell lines |
| GSE65161 |
Mediator kinase inhibition further activates super-enhancer-associated genes in AML |
|
| Relations |
| BioSample |
SAMN03290598 |
| SRA |
SRX849396 |
