|Public on Nov 16, 2020
|BT12 cells, IPAc, Solv_D, ChIP-seq
|Atypical Teratoid/Rhabdoid Tumor
|cell line: BT12
chip antibody: H3K27ac (Active Motif 39135)
|BT12 cells were treated with 100nM mithramycin for 8-hours or 18-hours continous treatment then collected.
|BT12 cells were grown in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Gemini Bio-Products), 2 mM L-glutamine (Invitrogen), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen). Cell lines were maintained at less than 80% confluency at 37°C and 5% CO2 and confirmed to be mycoplasma negative every 6-months.
|Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Sequencing libraries were prepared using the Kapa Biosystems Hyper Prep kit for the Illumina platform.
|Illumina NovaSeq 6000
|Libraries were sequenced using 2x50 bp sequencing on the Illumina NovaSeq platform at the Van Andel Research Institute. Data were demultplexed using bcl2fastq v2.20.0.
Reads were aligned using bwa mem, duplicate marked with samblaster, and filtered and converted to BAM format using samtools. Aligned reads were retained if they were aligned and had a minimum mapping quality of 20 (e.g. -F 4 -q 20 flags in samtools). BAMs were ingested into R (v3.6.1) and processed using csaw (v1.18.0). Briefly, a 150 bp sliding window with a 50 bp step-size was used to summarize the read counts with a maximum fragment size set to 800 bp. Next, the background was estimated using a 5kb sliding window where reads were binned and summarized. Regions were excluded if they overlapped known blacklist regions in hg19. Regions having signal greater than log2(3) fold-change over background were retained for differential binding analysis. The first principal component was regressed out of the data due to a batch effect being present prior to downstream analysis. Differential binding analysis was carried out using csaw and edgeR, fitting a quasi-likelihood (QL) negative binomial generalized log-linear model that estimates the prior QL dispersion distribution robustly. Differences were tested using an anova-like test or individual contrasts to generate initial differentially bound regions (DBR). Windows were merged with a maximum width of 5kb and a tolerance of 100bp between adjacent windows to consider being combined. Next, p-values were combined across clustered sites using Simes’ method to control the cluster false discovery rate as implemented in the combineTests function in csaw. Finally, clustered DBRs were defined as having a q < 0.05.
Supplementary_files_format_and_content: Bigwig files for raw and lambda corrected (pc1_corrected) peaks from all samples.
|Sep 13, 2019
|Last update date
|Nov 16, 2020
|Children's Hospital of Philadelphia
|3501 Civic Center Blvd
|SWI/SNF inhibition leads to epigenetic reprogramming in rhabdoid tumor [ChIP-seq]
|SWI/SNF inhibition leads to epigenetic reprogramming in rhabdoid tumor