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| Status |
Public on Aug 24, 2023 |
| Title |
CUT_CD34_WT_ETV6_rep1 |
| Sample type |
SRA |
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| Source name |
Bone Marrow
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Bone Marrow
cell type: CD34
genotype: Etv6+/+
treatment: Cut&Run antibody
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CUT&RUN was performed as previously described3,4. 1x10^6 HPC5 cells were harvested from culture and 50-100,00 LSK cells were isolated from mouse bone marrow as previously described. After three rinses each with 1-ml wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and protease inhibitors) followed by 3 min 600 g spin at RT, cells in 1 ml wash buffer were captured by Concanavalin A-coated magnetic beads (10 µl per million cells; Bangs laboratories, Inc.) at RT for 10 min with constant rotation. Upon three more washes, captured cells were permeabilized in 100 to 300 µl of the same wash buffer plus 0.05% digitonin and 2 mM EDTA with simultaneous antibody binding (purified anti-ETV6 antibody and corresponding amounts of IgG control) at 4°C overnight. Permeabilized cells/nuclei retained on the beads were washed 3 times with the same digitonin-wash buffer before incubation with 150 µl of Protein A-fused Micrococcal Nuclease (pA-MNase; 700 ng/ml) at 4°C for 1 h. After washing away unbound pA-MNase, antibody-associated protein-DNA complex was released from chromatin by adding CaCl2 to digitonin-wash buffer for a final 2 mM CaCl2 concentration and incubated at 0°C (i.e., ice-water bath) for 30 min. Equal volume of 2x stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% digitonin, 100 µg/ml RNase A, 50 µg/ml glycogen) containing spike-in yeast DNA (20 pg/ml) was then added to the reaction with an additional 30-min incubation at 37°C. The released DNA was isolated by proteinase K treatment and phenol/chloroform extraction and ethanol precipitation4. NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) was used to prepare CUT&RUN libraries. Barcoded libraries were sequenced using NextSeq 500 High Output.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
FASTQ files were mapped to mm10 by using BWA-MEM (version 0.7.16a). Reads that could not be uniquely mapped to the mouse genome were removed by SAMtools (version 0.17). Peaks were called using MACS2 (version 2.1.1). bigWig files were generated using DeepTools (version 3.2.0). Footprint analysis was performed by using CUT&RUNTools. Downstream analysis for binding site distribution and peak annotation were performed using R Bioconductor packages DiffBind7, ChIPseeker8, and clusterProflier9,10.
Assembly: mm10, hg38
Supplementary files format and content: bigwig tracks to be loaded in a genome browser
Library strategy: CUT&RUN
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| Submission date |
Sep 18, 2022 |
| Last update date |
Feb 28, 2024 |
| Contact name |
Kim Nichols |
| E-mail(s) |
kim.nichols@stjude.org
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| Organization name |
St. Jude Children's Research Hospital
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| Department |
Oncology
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| Lab |
Nichols Lab
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| Street address |
262 Danny Thomas Pl
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| City |
Memphis |
| State/province |
TN |
| ZIP/Postal code |
38105 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE213594 |
ETV6 Represses Inflammatory Response Genes and Regulates HSPC Function During Stress Hematopoiesis in Mice [CUT&RUN] |
| GSE213597 |
ETV6 Represses Inflammatory Response Genes and Regulates HSPC Function During Stress Hematopoiesis in Mice |
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| Relations |
| BioSample |
SAMN30877640 |
| SRA |
SRX17596861 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6589568_CUT_CD34_WT_ETV6_rep1.rmdup.uq.bw |
30.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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