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Sample GSM8133861

Query DataSets for GSM8133861

Status

Public on Jun 02, 2024

Title

H3K27ac, U2OS ASH1L knockout cells, no UV, biol rep2, input

Sample type

SRA

Source name

U2OS

Organism

Homo sapiens

Characteristics

cell line: U2OS
cell type: ASH1L knockout
treatment: no UV
antibody: none (input control)

Treatment protocol

Cells grown to 80-90% confluency were left untreated (non-UV) or were UV-C irradiated at 20 J/m2 and left to recover for 3h.

Growth protocol

Low glucose DMEM, 10% fetal calf serum. Cells were cultured at 37C at 5% CO2.

Extracted molecule

genomic DNA

Extraction protocol

Non-UV or 3h-post UV cells were crosslinked with 1% formaldehyde for 10 minutes. Quenching and lysis was followed by sonication (BioRuptor Plus) for 20 cycles (30 sec on/off).
To remove UV-induced DNA lesions with the potential to inhibit sequencing, immunoprecipitated, de-crosslinked fragments were incubated with PreCR Repair mix (New England BioLabs, M0309) for 15 min at 37°C, then re-purified, quantified by Qubit (Invitrogen, Q33238) and sent to the Functional Genomics Center Zurich for final library preparation with the NEB Next Ultra Kit (New England BioLabs, E7645). Libraries were sequenced as 100 bp single-end reads on the NovaSeq 6000.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NovaSeq 6000

Description

H3K27ac_ALKO_noUV_IN2

Data processing

Raw reads were trimmed to remove reads shorter than 99 base pairs and crop reads exceeding 101 bp (trimmomatic v.0.39), then aligned (bwa mem v.0.7.17-r1188) to the human reference genome build GCA hg38 lacking chrY but including non-canonical contigs. Alignments were filtered to exclude reads with mapping quality scores below 30 (bedtools v.2.29.2), PCR and optical duplicates (Picard MarkDuplicates, v.2.23.8), ENCODE-blacklisted regions, non-canonical contigs, and unmapped or secondary alignments (and for paired-end top-up reads, unpaired reads) (samtools v.1.7).
Filtered alignments were used to call peaks (macs2 v.2.2.7.1) from treatment (IP) over input control tagAlign files, where input control replicates were pooled together for all replicates and, following the ENCODE ChIP-seq processing guidelines, subsampled to 200 million reads for the H3K27ac ChIP-seq assays from wildtype cells.
Peaks were called with default parameters of macs2 using a cutoff p-value of 1e-2; broad peaks were called for H3K4me1 and narrow peaks for H3K27ac ChIP-seq. Peaks were used to verify replicabiity with idr (v.2.0.4.2). Only robust peaks (those shared across at least two of three biological duplicates) were retained in downstream analyses. The top quartile of robust peaks were determined by rank ordering peaks by signal strength. Fold enrichment (bigWig) tracks were generated of IP signal over input control.
Assembly: hg38
Supplementary files format and content: BED files: gzipped broadPeak (for H3K4me1) or narrowPeak (for H3K27ac) for each biological replicate.
Supplementary files format and content: bigWig files: Fold change (IP over IN) for each biological replicate.

Submission date

Mar 07, 2024

Last update date

Jun 02, 2024

Contact name

Michelle Yancoskie

Organization name

Institute of Veterinary Pharmacology and Toxicology, University of Zurich

Department

Vetsuisse Faculty

Lab

Naegeli Lab