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| Status |
Public on Apr 28, 2024 |
| Title |
U2OS_SOX2FL_day4_bio rep2 |
| Sample type |
SRA |
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| Source name |
cell line
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| Organism |
Homo sapiens |
| Characteristics |
tissue: cell line
cell line: U2OS
cell type: Human Osteosarcoma
genotype: Tet-on SOX2
treatment: 500ng/ml doxycycline for 4 days
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| Treatment protocol |
500ng/ml Doxycycline for 4 days
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| Growth protocol |
U2OS cells were cultured with 10% FBS with DMEM media
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Stable U2OS cell lines with Tet-on expression were induced with 500ng/ul Doxycycline for 4 days and processed with ChIP-seq protocol. 1x10^7 cells were cross-linked by 1% PFA for 10 min at room temperature and quenched by 125 mM glycine for 5 min. Pelleted cells were first lysed with NP40 buffer (50 mM Tris-HCl, pH 8.1, 150 mM NaCl, 5 mM EDTA, 0.5% NP40), and then resuspended with sonication buffer (20mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) for chromatin sonication by Bioruptor sonicator (Diagenode) at High output for 15 cycles (30s on, 30s off) at 4°C. Samples were centrifuged at 12000g for 10 min at 4°C and the supernatant was collected for IP. 4 μg ChIP-grade Flag antibody (Sigma, A01868-40) was added to the collected sample and incubated for 4 hours at 4°C with gentle shaking, followed by incubation of protein-G magnetic beads rocking at 4 °C overnight. Immunopreciated DNA was purified by Zymo DNA purification kit. Library construction was prepared using the NextEra NEB library prep kit for illumina (NEB, e7370) following the manufacturer’s protocol. DNA was size selected at 300-700 bp by electrophoresis and the sequencing was performed on the Illumina NOVA-seq 6000 Genome Analyzer (pair-end, 150bp).
ATAC-seq libraries were prepared using the library preparing kit (Vazyme, TD501). Transposed DNA was amplified for 10 cycles, and the library DNA was further size selected with VAHTSTM DNA Clean Beads (Vazyme, N411) to the range of 150bp - 700bp./
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Nova-seq output paired ChIP-seq raw reads quality control performed with FastQC (version 0.10.1). ChIP-seq raw reads were trimmed by trim_galore function with --paired mode and default parameters. ChIP-seq reads were aligned to hg38 genome with bowtie2 v2.4.4 in the following options: bowtie2 -t -q -p 10 -N 1 -L 25 -X 700. Resulted sam files were converted to bam with samtools (v.0.1.18). ChIP-seq bam files of the two replicates were called peaks individually using MACS2 with default parameters. For each group, peak files of the two replicates were used to call peaks by the IDR method with parameter: idr --input-file-type narrowPeak --rank p.value -soft-idr-threshold 0.05 The bigwig files of ChIP-seq data were generated using deeptools (v.2.4.1) with the following configurations: bamCoverage -of bigwig --binSize 50 —normalizeUsing RPKM --ignoreDuplicates. Assembly: hg38 Supplementary files format and content: Bigwig -- visualization of track information Supplementary files format and content: NarrowPeak -- Binding Sites for ChIP-seq/ Opening Site for ATAC-seq
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| Submission date |
Dec 28, 2023 |
| Last update date |
Apr 29, 2024 |
| Contact name |
Wulan Deng |
| E-mail(s) |
denglabpku@outlook.com
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| Organization name |
Peking University
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| Department |
BIOPIC
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| Street address |
Beijing Haidian District Yiheyuan Road #5
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| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE203644 |
Mesoscale chromatin confinement facilitates target search of pioneer transcription factors in live cells [ChIP-seq] |
| GSE203650 |
Mesoscale chromatin confinement facilitates target search of pioneer transcription factors in live cells |
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| Relations |
| BioSample |
SAMN39182411 |
| SRA |
SRX23052730 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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