|
| Status |
Public on Dec 01, 2016 |
| Title |
KARPAS-422_EZH2i_8d_H3K9me3 |
| Sample type |
SRA |
| |
|
| Source name |
Diffuse large B cell lymphoma
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: KARPAS-422
cell type: Diffuse large B cell lymphoma
treated with: EZH2i for 8 days
chip antibody: H3K9me3
chip antibody vendor: Active Motif
chip antibody cat. #: AM 39161
chip antibody lot #: Lot 2
|
| Growth protocol |
KARPAS-422 cell line was grown in Roswell Park Memorial Institute (RPMI) GlutaMAX medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. For each ChIP reaction, 30 ug of chromatin was precleared with protein A agarose beads (Invitrogen). ChIP reactions were set up using precleared chromatin and antibodies and incubated overnight at 4 C. Protein A agarose beads were added and incubation at 4 C was continued for another 3 hr. Immune complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on HiSeq 2500 (50 nt reads, single end).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
50-nucleotide sequence reads were aligned to the hg19 genome using the BWA algorithm with default settings. Only reads that passed Illumina’s purity filter, aligned with no more than 2 mismatches and mapped uniquely to the genome, were used in subsequent analyses.
The aligned read files were sorted and duplicates removed using sort and rmdup functions from samtools version 0.1.18 (Li et al., 2009).
Peak calling was done with the SICER-rb script (SICER v1.1; without Input file; using gap=600).
Genome_build: hg19
Supplementary_files_format_and_content: SICER scoreisland interval data for each sample in BED format.
|
| |
|
| Submission date |
Nov 20, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Barbara M Bryant |
| E-mail(s) |
barbara.bryant@constellationpharma.com
|
| Organization name |
Constellation Pharmaceuticals
|
| Street address |
215 First Street
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE63494 |
A ChIP-seq spike-in method enables detection of global histone modification changes across the genome |
|
| Relations |
| BioSample |
SAMN03202158 |
| SRA |
SRX764386 |
