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Sample GSM7695927

Query DataSets for GSM7695927

Status

Public on Nov 02, 2023

Title

MV411_ATACseq_1h_DMSO_rep1

Sample type

SRA

Source name

MV411

Organism

Homo sapiens

Characteristics

cell line: MV411
cell type: AML
genotype: wt
treatment: 0.1% DMSO, 1h

Growth protocol

Cells were maintained in RPMI medium consisting of RPMI 1640 with L-glutamine (Corning MT 10-040-CV), 10% fetal bovine serum (Fisher Scientific 16-000-044) and Penicillin-Streptomycin (Thermo Fisher 15140163).

Extracted molecule

other

Extraction protocol

ATAC-seq was performed as described in Grandi et al., (PMID: 35478247) with some modifications. Briefly 100,000 cells were mixed with 10,000 S2 Drosophila cells, and pelleted at 500g for 5 minutes at 4°C. Cells were washed with 100 µL ice cold DPBS (Corning 21-031-CV), and pelleted at 500g for 5 minutes at 4°C. The cell pellet was resuspended in 50 µL of ATAC-seq Lysis Buffer (0.1% NP40, 0.1% Tween-20, 0.01% digitonin, 10 mM Tris–HCl pH 7.5, 10 mM NaCl and 3 mM MgCl2) and incubated on ice for 3 minutes. Then 1 mL of ATAC-seq Wash Buffer (0.1% Tween-20, 10 mM Tris–HCl pH 7.5, 10 mM NaCl and 3 mM MgCl2) was added to dilute the lysis reagents, and nuclei were pelleted by spinning at 500g for 19 minutes at 4°C. Nuclei were then resuspended in 50 µL Transposition Mix (1X Tagment DNA Buffer, 0.33X DPBS, 0.01% digitonin, 0.1% Tween-20) treated with 3 µL TDE1 Tagment DNA Enzyme (Illumina 20034197). The reaction was incubated at 37°C for 30 minutes shaking at 1,000 rpm. Tagmented DNA was subsequently purified using the DNA Clean and Concentrator-5 Kit (Zymo Research, cat. no. D4014).
Purified samples were combined with NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs M01541) for amplification. As described in Grandi et al., (PMID: 35478247), custom primers were used to incorporate Illumina adaptors and index sequences into sample fragments.

Library strategy

ATAC-seq

Library source

other

Library selection

other

Instrument model

NextSeq 550

Data processing

All custom scripts described here are accessible at zenodo (doi:10.5281/zenodo.5519915).
Reads were trimmed for a minimum quality score of 20 using a custom script trim_and_filter_PE. Cutadapt 1.14 was used to remove adaptor sequences and low-quality reads.
In order to identify spike-in reads, read pairs were next aligned to the D. melanogaster genome (dm6) using bowtie 1.2.2 (-k1 -v2 -X1000, --best --p 5 --allow-contain).
All reads that failed to align to the spike genome were subsequently aligned to the H. sapiens genome (hg38) using bowtie 1.2.2 (-k1 -v2 -X1000 --best -p 5 -S --allow-contain).
Fragments were deduplicated and filtered to retain unique reads between 10 and 150 bp, representing regions of accessible chromatin, which were then converted to bedGraph format using the custom script extract_fragments.pl. As spike returns were consistently elevated in BRM014 and AU-5330 treated conditions, samples were spike-normalized using the custom script normalize_bedGraph.pl. As the replicate samples were highly correlated across ATAC-seq peaks, biological replicates were merged using the custom scripts bedgraphs2stdBedGraph.pl. Data was binned in 50 bp windows to generate bedGraph files for UCSC Genome Browser visualization and downstream analysis.
Assembly: hg38
Supplementary files format and content: All files with the .bw suffix are normalized BigWig files containing combined data from all replicates of the indicated treatment condition.

Submission date

Aug 07, 2023

Last update date

Nov 02, 2023

Contact name

Karen Adelman

E-mail(s)

karen_adelman@hms.harvard.edu

Organization name

Harvard Medical School

Department

Biological Chemistry and Molecular Pharmacology