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| Status |
Public on May 25, 2022 |
| Title |
mESC ATAC-seq rep2 |
| Sample type |
SRA |
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| Source name |
OG2 mESC
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| Organism |
Mus musculus |
| Characteristics |
cell line: OG2
cell type: mESC
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| Growth protocol |
ciTotiSCs were cultured in KSR basal medium composed of KO DMEM, 5% KSR (Gibco, 10828010), CDL (CD lipid concentrate, 500X, Gibco, 11905-031), 1% N2 (ThermoFisher, A1370701), 1X L-glutaMAX, 1X penicillin-streptomycin, 1X non-essential amino acids (NEAA), 1X sodium pyruvate, 55 μM 2-mercaptoethanol and 1000 U/mL mouse leukemia inhibitory factor (mLIF), 50 ng/ml Sodium L-ascorbyl-2-phosphate (Selleck, S5115) supplemented with 2.5 μM 1-Azakenpaullone (Selleck, S7193), 0.5 μM WS6 (Selleck, S7442) and 0.2 μM TTNPB (Selleck, S4627). Cells were passaged every 2-3 days using 0.05% Trypsin-EDTA at a ratio of 1:3-1:5 on inactivated MEF feeder layers.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, after cells were collected by flow cytometry as described before, 100,000 cells were centrifuged at 500 rcf for 5 min in a 4 °C pre-chilled fixed-angle centrifuge and washed by ice-cold PBS. After centrifugation, supernatant was removed with two pipetting steps to avoid the cell pellet. Cell pellets were then resuspended in 50 μl of ATAC-seq RSB containing 0.1% IGEPAL-630, 0.1% Tween-20, and 0.01% digitonin and incubated on ice for 10 min. After lysis, 1 ml of ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin) was added, and the tubes were inverted for six times to mix. Nuclei were then centrifuged for 10 min at 500 rcf. in a 4 °C pre-chilled fixed-angle centrifuge.
Supernatant was removed and nuclei were then incubated with the Tn5 transposome and tagmentation buffer at 37 °C for 30 min (Novaprotein, N248). After the tagmentation, the stop buffer was added directly into the reaction to end the tagmentation. Reactions were cleaned up with 2.2x VAHTS DNA Clean Beads (Vazyme, N411). PCR was performed to amplify the library for 8 cycles by KAPA HiFi PCR Kit (Kapa Biosystems, kk2102) using the following PCR conditions: 72 °C for 5 min; 98 °C for 3mins; and thermocycling at 98 °C for 20 s, 63 °C for 30 s and 72 °C for 3 min. After the PCR reaction, libraries were purified with the 1.8x VAHTS DNA Clean Beads.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
cut adapter using python cutadapt version 1.18 parameter -q20 --quality-base=33 adapters detected using first 1million reads
reads aligned to mm10 genome using bowtie2 version 2.3.3.1 with parameters -X2000 -t -1 -N1 -L 25 --no-mixed --no-discordant --threads 16
using samtools(version 1.6) to filter out low mapping quality reads samtools view -F 1804 -f 2 -q 30 -@ {threads} -u {input.bam} | samtools sort -@ {threads} -m 2G -n /dev/stdin -o {output.bam}
using samtools to sort reads by coordinate and fixmate samtools fixmate -@ {threads} -r {input.bam} {output.fixmate} samtools view -F 1804 -f 2 -@ {threads} -u {output.fixmate} |\ samtools sort -@ {threads} -m 2G /dev/stdin -o {output.bam}
using picard(version 2.20.4) and samtools to remove duplicated reads java -Xmx10G -Djava.io.tmpdir={params.tempdir} \ -jar /applications/picard_2.20.4/picard.jar MarkDuplicates \ INPUT={input.bam} \ OUTPUT={output.marked} \ METRICS_FILE={output.metrics} \ VALIDATION_STRINGENCY=LENIENT \ ASSUME_SORTED=true \ REMOVE_DUPLICATES=false samtools view -F 1804 -f 2 -@ 5 -b -u {output.marked} |\ samtools sort -@ 5 -m 2G /dev/stdin -o {output.nodup} samtools index -@ 5 {output.nodup}
using bedtools(version 2.26.0) to filter out unmappable genome regions according to blacklist(https://github.com/Boyle-Lab/Blacklist/tree/master/lists/Blacklist_v1) bedtools intersect -v -abam {input.bam} -b {params.blacklist} |\ samtools view -F 1804 -f 2 -@ {threads} -S -h -b |\ samtools sort -@ {threads} -m 2G /dev/stdin -o {output.bam} samtools index -@ {threads} {output.bam}
using bedtools to shift each end of mate pair 5bp toward mate pair center, and make a bed file with the shifted end as center, and extend 125bp to each side, that marks the total 250bp region we considered accessible
convert such a bed file to bigWig file mentioned below using bedtools genomecov
using macs2 (version 2.2.7.1) to callpeak with parameters: --bdg -g mm --nomodel --call-summits this produced narrowPeak file mentioned below
Genome_build: mm10
Supplementary_files_format_and_content: bigwig file of two replicates of ciTotiSC and mESC and narrowPeak file of two replicates of ciTotiSC and mESC
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| Submission date |
Sep 29, 2021 |
| Last update date |
Jun 27, 2022 |
| Contact name |
Pengcheng Tan |
| E-mail(s) |
tpchengbjhd@126.com
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| Organization name |
Tsinghua University
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| Street address |
30 Shuangqing Rd
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| City |
Haidian |
| ZIP/Postal code |
100084 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE184999 |
Induction of mouse totipotent stem cells by a defined chemical cocktail [ATAC-seq] |
| GSE185005 |
Induction of mouse totipotent stem cells by a defined chemical cocktail |
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| Relations |
| BioSample |
SAMN21898829 |
| SRA |
SRX12403356 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5603032_ATAC-mESC-2.ext125.rpkm.bw |
73.9 Mb |
(ftp)(http) |
BW |
| GSM5603032_ATAC-mESC-2.narrowPeak.gz |
1.2 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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