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| Status |
Public on Oct 20, 2021 |
| Title |
CnT-BS_H3K27me3_100K |
| Sample type |
SRA |
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| Source name |
E14 mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14 mouse embryonic stem cells
chip antibody: anti-H3K27me3 (CST #9733S)
cell count: 100,000
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| Growth protocol |
Mouse E14 embryonic stem cells (129/Ola background) were maintained in Glasgow Minimum Essential Medium (GMEM, Sigma) containing 15% fetal bovine serum, supplemented with 1× Pen-Strep (Gibco), 2 mM Glutamax (Gibco), 50 µM β-mercaptoethanol (Gibco), 0.1 mM nonessential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), and Leukemia Inhibitory Factor (LIF, 1000U/ml, Millipore).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CUT&Tag was performed according to previously published protocol (Kaya-Okur et al., 2019) with slight modification, namely using pA-Tn5 transposome assembled with methylated adapter instead. Briefly, harvested cells were washed and then bound to concanavalin A-coated magnetic beads. The cells were permeabilized and incubated with target-specific primary antibody and then with secondary antibody, followed by incubation with pA-Tn5 transposome and tagmentation at target-binding sites. The tagmented DNA was extracted with Qiagen MinElute PCR Purification Kit.
In CUT&Tag-BS, the purified CUT&Tag-DNA was subsequently subjected to tagmentation-based bisulfite sequencing library preparation, which includes an oligonucleotide replacement and gap repair step to ensure that each DNA strand is covalently attached to methylated adapters on both ends, followed by bisulfite conversion and PCR amplification to generate sequencing library. While for non-bisulfite-treated CUT&Tag control, the bisulfite conversion step was removed in library preparation.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
unified-H3K27me3.peaks.bed.gz
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| Data processing |
Library strategy: CUT&Tag-BS
Filtering and mapping: Raw reads were filtered to exclude pairs with average base quality <20. Adapters removed by Cutadapt v1.12 with parameters "-a CTGTCTCTTATACAC -A CTGTCTCTTATACAC -O 5 -q 0 -m 20 -p", then reads were filtered to exclude fragments with length <30bp. Filtered, trimmed read pairs were mapped against mm10 via Bismark v0.23.0 with "-X 2000" using Bowtie2 v2.3.0 as the underlying mapper. Duplicate mapped fragments were removed by the Bismark v0.23.0 deduplicate_bismark tool with parameter "-p".
Peak calls: HOMER v4.10.3 was used for peak calls with the makeTagDirectory function (parameters "-format sam -read1 -keepAll -fragLength 200") followed by the findPeaks function (parameters "-size 500 -minDist 1000 -L 0 -region" for H3K4me1 and "-size 1000 -minDist 2500 -L 0 -region" for H3K9me3 and H3K27me3). Peaks overlapping mm10 blacklist regions were discarded. Unified peak sets were defined as the union of peak calls (per mark) via BEDtools v2.29.2 merge.
Signal tracks: Coverage tracks for genome browser views were generated by converting paired-end hits to a single fragment by BEDtools v2.29.2 bamtobed with the "-bedpe" option, BEDtools v2.29.2 genomecov to generate bedGraph format, then UCSC utility bedGraphToBigWig to convert to bigWig format.
Collecting methylation counts: The Bismark v0.23.0 bismark_methylation_extractor tool was run with parameters "-p --ignore_r2 9 --comprehensive --mbias_off --bedGraph --cytosine_report".
Percent methylation tracks: For visualization purposes, the methylated and unmethylated cytosine counts from both strands (in the CpG context output from Bismark v0.23.0 bismark_methylation_extractor) were collapsed to calculate percent methylation per genomic CpG. Files were converted from variableStep WIG format to bigWig format by UCSC utility wigToBigWig.
Genome_build: mm10
Supplementary_files_format_and_content: percent methylation per CpG in bigWig format, coverage in bigWig format, peaks in BED format, matrix of methylated and total cytosine base counts per CpG in tab-delimited text format
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| Submission date |
Sep 16, 2021 |
| Last update date |
Oct 20, 2021 |
| Contact name |
ruifang li |
| E-mail(s) |
lir4@niehs.nih.gov
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| Organization name |
NIEHS
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| Street address |
111 T.W. Alexander Drive
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| City |
RTP |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (1) |
| GSE179266 |
CUT&Tag-BS: an efficient and low-cost method for simultaneous profiling of histone modification and DNA methylation |
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| Relations |
| BioSample |
SAMN21462753 |
| SRA |
SRX12209245 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5583570_CnT-BS_H3K27me3_100K.CpG-pctMeth.bigWig |
16.9 Mb |
(ftp)(http) |
BIGWIG |
| GSM5583570_CnT-BS_H3K27me3_100K.coverage.bigWig |
69.8 Mb |
(ftp)(http) |
BIGWIG |
| GSM5583570_CnT-BS_H3K27me3_100K.peaks.bed.gz |
182.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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