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Sample GSM3453123

Query DataSets for GSM3453123

Status

Public on Nov 18, 2019

Title

ATAC-seq.Sorted-no2DG_rep1

Sample type

SRA

Source name

mESCs

Organism

Mus musculus

Characteristics

tissue: Mouse embryonic stem cells (mESCs)
condition: None 2DG treated, GFP negative, FACS-sorted

Treatment protocol

In ATAC-seq, Culture media was supplemented with 4mM 2-DG for 4 days to induce NELFA for reporter mESC, and 0.4ug/mL of Doxycycline for Dox-inducible NELFA mESCs.

Growth protocol

mESCs were cultured on gelatin coated cell culture dishes with knockout DMEM supplemented with serum, LIF and 2i.

Extracted molecule

genomic DNA

Extraction protocol

ATAC-Seq: 2-DG treated and Dox-induced mESCs were FACs sorted to enrich for both GFP- positive and -negative populations. Collected mESCs were lyzed in hypotonic buffer containing NP-40. Isolated cell nuclei were then subjected tagmentation with Illumina Tn5 transposase before PCR amplification with NEBNext 2X PCR mastermix and Illumina Nextera indexed primers.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

GSE113671_2DGminus_ATAC.bw

Data processing

Reads were trimmed to remove the adapter sequences and low quality bases using Trim_Galore (v0.4.2_dev).
For ATAC-seq, paired-end raw sequencing reads were trimmed with Trim Galore to remove adapters and trim low quality reads. Cleaned reads were then mapped to the mouse reference genome mm10 with Bowtie2 (v.2.2.9) with the following parameters: --N 1 --L 25 --X 2000 --no-mixed --no-discordant. Mapped reads were sorted and PCR duplicates were removed using SAMtools(v1.4). Biological replicate level ATAC-seq differential peaks were called between conditions with MACS2 'callpeak' (--B --nomodel --shift -100 --extsize 200), generating replicate-level bedgraph files, followed by the 'bdgdiff' subcommand (--l 500 --g 250) to call 'differential peaks'. Subsequently, high quality overlapping peaks between two replicates were selected using Bedtools 'intersect' (-u -f 0.5 'F 0.5) for condition specific peaks. For coverage bigwig file generation, replicate-merged bedgraph files were generated using MACS2 'callpeak' (--B 'SPMR 'nomodel --shift -100 --extsize 200 't replicate1.bam replicate2.bam), which were subsequently converted to bigwig files using 'bedGraphToBigWig' from Kent informatics. To detect the chromatin accessibility across repetitive element regions, mapped reads were further filtered with SAMtools (view 'q 10) to remove low-quality multiple-mapping reads, and coverage bigwig files were generated as mentioned.
Genome_build: mm10
Supplementary_files_format_and_content: BIGWIG files contain theATAC-seq coverage; EXCEL file contains condition specific ATAC gained and lost regions.

Submission date

Oct 31, 2018

Last update date

Nov 18, 2019

Contact name

Zhenhua hu

E-mail(s)

zhhu@imcb.a-star.edu.sg

Phone

65869644

Organization name

IMCB, A*STAR