|
| Status |
Public on Jan 30, 2023 |
| Title |
OCIAML3-NPM1c-FKBP_ChIP_CDK9_DMSO_24h_REP1_220623_GATCAG_S9 |
| Sample type |
SRA |
| |
|
| Source name |
Derivative of cell line OCI-AML3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: OCIAML-3
antibody: CDK9
antibody manufacturer: Santa Cruz
antibody catalog number: 13130
agent: DMSO
timepoint: 24 hours
|
| Treatment protocol |
Treatment regimens indicated for each sample.
|
| Growth protocol |
Human cell lines were cultured in RPMI with 10% FBS.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with 1% methanol-free formaldehyde (ThermoFisher) for 7-10 min at room temperature, followed by quenching with 100 mM Tris pH8.0 and 25 mM Glycine, and the cells were lysed in SDS buffer. Cytoplasm was lysed using 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM EDTA, 1% SDS for 10 min and nuclei were precipitated by centrifugation at 10,000´g. Nuclei were resuspended in 66 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM EDTA, 1.7% Triton X-100, 0.5% SDS and sheared using E100S (Covaris) to chromatin fragments of 200-400 base-pair DNA size.
1-10 ng of DNA was used in preparation of Illumina-compatible libraries using ThruPlex DNA kit (Rubicon Genomics)
Illumina Next Gen Sequencing NextSeq platform (Illumina, San Diego, CA) was used to obtain 1-5´107 unique sequencing paired-end tags
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ChIPseq_MACS2_peak_bedtools_coverage_diffrpk_OCIAML3-NPM1c-FKBP_Supp_S1.xlsx
|
| Data processing |
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the human(Gencode GRCh38/hg38) genome using STAR (v2.7.5a; params --alignIntronMax 1 --alignEndsType EndToEnd --alignM atesGapMax 2000 for ChIPseq analysis), sorted and duplicates marked/removed with picard pipeline tools (v2.9.4).
Final “deduped” .BAM files were indexed using SAMtools (v1.95).
ChIP-seq data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75) and ngs.plot (pileup heatmaps). ChIPseq peaks were called using MACS2 with appropriate input samples used as controls.
Peak-associated signals were assessed using the bedtools coverage function. Single peaks were chosen as representative of change in gene signal, using the peak overlapping the gene body which showed the greatest differential signal between treated and control ChIP samples. Signal values were normalized based on the ratio of hg38-aligning reads in treated and control samples, after deduplication.
Assembly: GRCh38/hg38
Supplementary files format and content: MACS2 peak interval signal summary (from bedtools coverage; Excel .xlsx format); Broad IGV (.tdf) files for visualization
|
| |
|
| Submission date |
Dec 01, 2022 |
| Last update date |
Jan 30, 2023 |
| Contact name |
Charlie Hatton |
| E-mail(s) |
Charles_Hatton@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Pediatric Oncology
|
| Lab |
Scott Armstrong/CPCT
|
| Street address |
450 Brookline Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE196476 |
Mutant NPM1 binds chromatin and directly regulates oncogenic transcription in acute myeloid leukemia [ChIP-seq] |
| GSE196480 |
Mutant NPM1 binds chromatin and directly regulates oncogenic transcription in acute myeloid leukemia |
|
| Relations |
| BioSample |
SAMN31976298 |
| SRA |
SRX18455086 |
