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| Status |
Public on Jun 20, 2024 |
| Title |
mESC_KAS-seq, rep2 |
| Sample type |
SRA |
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| Source name |
mESC
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| Organism |
Mus musculus |
| Characteristics |
cell line: mESC
cell type: Mouse embryonic stem cell
genotype: WT
treatment: No treatment
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| Treatment protocol |
Cells harvested from the culture dish or Fluorescence-activated cell sorting (FACS) cells were washed with DPBS and subsequently resuspended in 50 µL of ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin. After gentle pipetting, the suspension was incubated on ice for 3 minutes. The cells were then treated with 1 mL of cold ATAC-RSB containing 0.1% Tween-20 and centrifuged at 500 RCF for 5 minutes at 4ºC. Following a wash with 1 mL DPBS, the cells underwent N3-kethoxal labeling at 37 ºC with continuous agitation. The labeling process involved a 15-minute incubation in 5mM N3-kethoxal in PBS.
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| Growth protocol |
Murine embryonic stem (ES) cells were purchased from ATCC (CRL-1821) and were cultured in DMEM (Gibco 11995) supplemented with 10% (v/v) fetal bovine serum (Gibco), 1 mM L-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1% (v/v) nonessential amino acid stock (100x, Gibco), 1% penicillin/streptomycin stock (100x, Gibco), and 1,000 U/mL LIF (Millipore)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Then, 1 µg genomic DNA was suspended in 95 µL DNA elution buffer supplemented with 5 µL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma, 760749), 25 mM K3BO3, and incubated at 37 °C for 1.5 h while being gently shaken. Next, 5 µL RNase A (Thermo, 12091039) was added into the reaction mixture followed by incubation at 37 °C for 5 min. Biotinylated gDNA was then recovered by DNA Clean & Concentrator-5 kit (Zymo, D4013).
gDNA was suspended into 100 µL water and was fragmented to 150–350 bp size by using Bioruptor Pico at 30s-on/30s-off setting for 30 cycles; 5% of the fragmented DNA was saved as input, and the remaining 95% was used to enrich biotin-tagged DNA by incubation with 10 µL pre-washed Dynabeads MyOne Streptavidin C1 (Thermo, 65001) at room temperature for 15 min. The beads were washed, and DNA was eluted by heating the beads in 15 µL H2O at 95 °C for 10 min. Eluted DNA and its corresponding input were used for library construction by using Accel-NGS Methyl-seq DNA library kit (Swift, 30024).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
To align Opti-KAS-seq and KAS-seq data to the reference genome of interest, we utilized the trim_galore package to trim off low-quality sequence, adaptor sequence and primer sequence from single-end or paired-end raw fastq files
Subsequently, we employed bowtie2 to perform the read alignment of Opti-KAS-seq and KAS-seq data. Mapped reads in sam files from the aligners are sorted and converted to bam files using ‘samtools sort’, which are subsequently deduplicated using ‘picard MarkDuplicates’ (pair-end data) or ‘samtools rmdup’ (single-end data).
For single-end Opti-KAS-seq or KAS-seq data, mapped reads were extended to 150bp as default, irrespective of the initial sequencing data's read length. For paired-end KAS-seq data, the KAS-Analyzer incorporates a Python script, facilitating the merging of "properly paired" mapped reads into a singular interval
Peak calling for broad KAS-seq was executed using epic2, which identified broad peaks maintaining a false discovery rate (FDR) of 0.05 and a 1.5-fold change relative to the Input. Comprehensive quality control measures, such as library complexity metrics, inter-replicate correlation analyses, fingerprint plots, saturation analyses, genomic distribution of KAS-seq peaks, enrichment within gene-coding regions, and the Fraction of Reads in Peaks (FRiP) metric, were executed with the KAS-Analyzer package.
Deduplicated mapped KAS-seq reads were converted to bedGraph and bigWig using deeptools bamCoverage and UCSC tools.
Assembly: mm10
Supplementary files format and content: bed, peaks list.
Supplementary files format and content: bigWig, read density.
Library strategy: KAS-seq
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| Submission date |
Feb 21, 2024 |
| Last update date |
Jun 20, 2024 |
| Contact name |
Ruitu Lyu |
| E-mail(s) |
lvruitu@uchicago.edu
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| Organization name |
The University of Chicago
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| Department |
Chemistry
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| Lab |
Chuan He
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| Street address |
929 E 57th Street
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL16417 |
| Series (2) |
| GSE256229 |
Quantitative analysis of cis-regulatory elements in transcription with KAS-ATAC-seq [Mouse KAS-seq] |
| GSE256232 |
Quantitative analysis of cis-regulatory elements in transcription with KAS-ATAC-seq |
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| Relations |
| BioSample |
SAMN40017760 |
| SRA |
SRX23679636 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8091605_mESC_KAS-seq.rep2.ext150.bigWig |
289.7 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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