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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 28, 2024 |
| Title |
BxPC3, IgG, Replicate2, CUT&RUN |
| Sample type |
SRA |
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| Source name |
BxPC-3
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| Organism |
Homo sapiens |
| Characteristics |
cell line: BxPC-3
cell type: PDAC
cut&run antibody: IgG (EpiCypher 13-0042k)
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| Growth protocol |
BxPC-3 cells were cultured in RPMI-1640 (Thermo Fisher Scientific) supplemented with 10% Serum Plus-II (Thermo Fisher)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
We performed CUT&RUN in BxPC-3 cells using CUTANA ChIC/CUT&RUN Kit (EpiCypher) following the manufacturer’s protocol (Skene & Henikoff, 2017). In brief, 500,000 cells were trypsinized, washed, and bound to activated Concanavalin A-conjugated paramagnetic beads (EpiCypher). The cell-bead conjugates were then resuspended in recommended buffer with 0.5 μg antibody targeting the protein of interest: Anti-BPTF (ThermoFisher Scientific A300-973A), Anti-SRF (Cell Signaling Technology 5147S), Anti-H3K4me3 (EpiCypher 13-0041k) as positive control, and Anti-IgG (EpiCypher 13-0042k) as negative control. The suspensions were incubated overnight at 4 °C with gentle shaking. Then, the cell-bead conjugates were washed twice with the kit’s permeabilization buffer, and incubated with pAG-MNase (Epicypher 15-1016) for 10 min at room temperature. Subsequently, the unbound pAG-MNase was washed away, and the MNase was activated by adding CaCl2 to a final concentration of 2 mM. The reaction was stopped after the pAG-MNase was allowed to digest and release the antibody-bound chromatin for 2 hours at 4 °C. Finally, the released DNA was purified with the spin columns provided by the kit (EpiCypher).
Library preparation was performed using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs). The libraries were sequenced on a Illumina NextSeq 500 using 2x75bp reads.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
The fastq files were trimmed and quality-controlled with Trim Galore (version 0.6.10 with the default options in a pair-end mode. The resulting reads were aligned to hg38 using bowtie2 (version 2.4.4) with options --local --very-sensitive-local --no-unal --no-mixed --no-discordant --dovetail -I 10 -X 700 (Langmead & Salzberg, 2012). The alignment files were sorted by coordinate, then unpaired reads were removed, and indexed with samtools (version 1.14) (Danecek et al., 2021).
For reads visualization on genome tracks, the processed bam files were merged with samtools and visualized via the Integrative Genomics Viewer (version 2.16.1) (Robinson et al., 2011).
For peak identification, peaks and summits were called with MACS2 (version 2.2.7.1) using all three individual bam files for all replicates targeting the protein of interest (-t) and all three targeting IgG as controls (-c) with options --format BAMPE --gsize 2.7e9 --keep-dup all (Zhang et al., 2008).sortRegions descend (Ramirez et al., 2016).
Assembly: hg38
Supplementary files format and content: bigWig, narrowPeak
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| Submission date |
Feb 13, 2024 |
| Last update date |
Nov 28, 2024 |
| Contact name |
Xinhe Xue |
| E-mail(s) |
[email protected]
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| Organization name |
New York Genome Center/New York University
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| Street address |
101 Avenue of the Americas
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| City |
NEW YORK |
| State/province |
New York |
| ZIP/Postal code |
10013 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE255699 |
Paired CRISPR screens to map gene regulation in cis and trans (CUT&RUN) |
| GSE255701 |
Paired CRISPR screens to map gene regulation in cis and trans |
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| Relations |
| BioSample |
SAMN39938780 |
| SRA |
SRX23617902 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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