|
| Status |
Public on Feb 15, 2020 |
| Title |
HEK293_10kcells_KAS-IP.rep1 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293 cells
|
| Organism |
Homo sapiens |
| Characteristics |
ip method or chip antibody: N3-kethoxal biotin IP
genotype: Wild type
|
| Treatment protocol |
Cells were incubated in completed culture medium containing 1‰ N3-kethoxal and for 5-10 min at 37 ˚C, 5% CO2.
|
| Growth protocol |
HEK293T cells were cultured in DMEM (Gibco 11995) supplemented with 10% (v/v) fetal bovine serum (Gibco), 1% penicillin and streptomycin (Gibco) and grown at 37 ˚C with 5% CO2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were harvested and genomic DNA (gDNA) was isolated from cells by PureLink genomic DNA mini kit. 1 µg genomic DNA was then suspended in 95 µL DNA elution buffer supplemented with 5 µL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma, 760749), and incubated at 37 ˚C for 1.5 h with gentle shake. 5 µL RNase A (Thermo, 12091039) was added into the reaction mixture followed by an incubation at 37 ˚C for 5 min. Biotinylated gDNA was then recovered by DNA Clean & Concentrator-5 kit (Zymo). gDNA was suspended into 100 µL water and subjected to sonication by using Bioruptor Pico at 30s-on/30s-off setting for 30 cycles. 5% of the fragmented DNA was saved as input, and the rest 95% was used to enrich biotin-tagged DNA by incubating 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).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Low-quality and adapter-containing reads were trimmed from KAS-seq raw data using trim-galore package under single-end mode, reads shorter than 50 bp were removed.
Trimmed reads were aligned to reference genome (hg19 for HEK293T cells or mm10 for mESC) using bowtie2 (v2.3.3.1) under default parameters. Mapped sam files were subsequently converted and sorted to bam files using samtools sort (v1.9).
Duplicates were removed using samtools rmdup (v1.9) to get the unique mapped reads. The unique and monoclonal mapped reads were extended to 150 bp to match the average length of insert DNA fragments of the KAS-seq libraries.
Bam files were converted to bed files and bedGraph files using bedtools. BedGraph files were then converted to bigWig files using bedgraphtobigwig from UCSC pre-compiled utilities. BedGraph files were used for visualization at UCSC genome browser and bigWig files were used to calculate tag density under 50-bp resolution.
We used MACS2 to call all reported KAS-seq peaks in this manuscript. Based on the KAS-seq profile on UCSC genome browser, most KAS-seq peaks on gene body are very broad. Therefore, MACS2 was run using broad peaks-call mode under default parameters, except for --broad-cutoff 0.1 and –qvalue 0.01.
hg19&mm10
KAS-seq tag density files(bigWig)&Peaks files(bed)
|
| |
|
| Submission date |
Oct 25, 2019 |
| Last update date |
Feb 15, 2020 |
| Contact name |
Ruitu Lyu |
| E-mail(s) |
lvruitu@uchicago.edu
|
| Organization name |
The University of Chicago
|
| Department |
Chemistry
|
| Lab |
Chuan He
|
| Street address |
929 E 57th Street
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE139420 |
In situ capture of global transcription dynamics and enhancer activity with high accuracy and sensitivity |
|
| Relations |
| BioSample |
SAMN13117339 |
| SRA |
SRX7059347 |
