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| Status |
Public on May 06, 2023 |
| Title |
CUTRUN_EryPro_ETV6_Rep1 |
| Sample type |
SRA |
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| Source name |
Cord Blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Cord Blood
cell line: Erythroid progenitor
cell type: Erythroid progenitor
genotype: pooled
antibody: ETV6
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CUT&RUN experiments were carried out as described with modifications. Briefly, 10,000 cells were captured with BioMagPlus Concanavalin A (Bang Cat. # BP531) for 10 min at room temperature. Permeabilization of cells and binding of primary antibodies were performed oner-night at 4℃. After washing away unbound antibody with wash buffer, protein A-MNase was added at a 1:100 ratio and incubated for 2 hours. Cells were washed again and placed in a 0℃-metal block. Then CaCl2 was added to a final concentration of 2 mM to activate protein A-MNase. The reaction was carried out for 30 min and stopped by addition of equal volume of 2XSTOP buffer. The protein-DNA complex was released by centrifugation. DNA was extracted by NEB PCR Cleanup Kit (NEB Cat. # T1030), followed by Qubit fluorometer and bioanalyzer quality control. Protein A-MNase was expressed and purified from BL21(DE) carrying pET-pA-MN. pET-pA-MN was obtained from addgene (Cat#:86973).
The CUT&RUN library preparation was performed with DNA library preparation kit (Vazyme, Cat. # ND607) following with the manufacture’s protocol with minor modifications. Briefly, the temperature dA-tailing was decreased to 50°C to avoid DNA melting, and the reaction time was increased to 1 hour. After adaptor ligation, 2X volume of DNA Clean beads (Vazyme, Cat. # N411) was added to the reaction to ensure high recovery efficiency of short fragments. After 15 cycles of PCR amplification, the reaction was cleaned up with 1.2X volume of DNA Clean beads. The final libraries were quantified using a VAHTS Library Quantification Kit (Vazyme, Cat. # NQ101) and a D1000 High Sensitivity DNA chip run on a Bioanalyzer 4250 system (Agilent). Two biological replicates per condition were sequenced in a Novaseq 6000 (S4, PE150, Illumina) by Novogene (Beijing).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw sequence data of CUT&RUN were trimmed to barcode using trim_galore (version 0.4.4_dev) and reads were aligned to hg19 version of human genome with bowtie2 (version 2.3.4.1).
Duplicated reads were removed through Picard tool (http://broadinstitute.github.io/picard/) and the low quality of reads (MAPQ < 30) were removed. Reads with the cutoff of inset size < 120bp were selected for transcription factors occupancy and 150-500bp for H3K27ac histone modification as previous studies.
Peaks were calling by SEACR with default parameter with IgG control
The differentiated binding sites of H3K27ac and GATA1 were calculated through diffbind R package with the following parameter (FDR < 0.05) (https://www.cruk.cam.ac.uk/core-facilities/bioinformatics-core/software/diffbind). Co-binding of transcription factors was performed by bedtools and R with 1 bp overlap. The bigwig/ bedgraph files were generated through bamCoverage (https://deeptools.readthedocs.io/en/develop/)
Co-binding of transcription factors was performed by bedtools and R with 1 bp overlap. The bigwig/ bedgraph files were generated through bamCoverage (https://deeptools.readthedocs.io/en/develop/) to visualize peaks in IGV (https://software.broadinstitute.org/software/igv/), and pyGenomeTrack (https://github.com/deeptools/pyGenomeTracks).
Assembly: hg19
Supplementary files format and content: bigWig
Library strategy: CUT&RUN
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| Submission date |
Oct 04, 2022 |
| Last update date |
May 06, 2023 |
| Contact name |
Dong Li |
| E-mail(s) |
lidongpaul@gmail.com
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| Organization name |
Peking University
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| Department |
College of Life Science
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| Lab |
Hsiang-Ying (Sherry) Lee
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| Street address |
No.5 Yiheyuan Road, Haidian District,
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| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE214805 |
Stage-specific erythroid cell three-dimensional chromatin architecture and transcription factors binding provide insight of human erythropoiesis [CUT&RUN] |
| GSE214811 |
Stage-specific erythroid cell three-dimensional chromatin architecture and transcription factors binding provide insight of human erythropoiesis |
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| Relations |
| BioSample |
SAMN30928912 |
| SRA |
SRX17639675 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6616155_CUTRUN_EryPro_ETV6_Rep1.bigwig |
48.4 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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