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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 03, 2025 |
| Title |
HAP1-KRAB, sgRNA targeted rs73156934-tagged CRE, H3K27me3 |
| Sample type |
SRA |
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| Source name |
HAP1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HAP1
strain: KRAB-blast stably expressed HAP1 cell
treatment: transduced with sgRNA targeted rs73156934-tagged CRE
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| Treatment protocol |
HAP1-sgURE and HAP1-sgNTC cells were generated by transduced KRAB-blast stably expressed HAP1 cell with sgRNA targeted rs73156934-tagged CRE or non-targeting control(NTC) .
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| Growth protocol |
HAP1 (Horizon Discovery) cells were cultured in IMDM (ThermoFisher) supplemented with 10% fetal bovine serum in 5% CO2 at 37°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Adherent cells were digested with 0.25% trypsin and collected in a 15mL tube containing serum-containing medium, followed by centrifugation and washing of the cell pellet with serum-free basal medium to obtain a single-cell suspension.
CUT&Tag experiments were performed using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903). Briefly, 1 × 10⁵ freshly cultured cells were centrifuged (500 × g, 5 min) and resuspended in 500 μL Wash Buffer. After adding 10 μL ConA Beads and incubating at room temperature for 10 min, the beads were collected with a magnetic rack. The beads were incubated with 50 μL of primary antibody (H3K27ac antibody, Cell Signaling Technology, 8173S; H3K4me1 antibody, Cell Signaling Technology, 5326S; H3K4me3 antibody, Abcam, 8898; H2AK119ub antibody, Cell Signaling Technology, 8240T; H3K27me3 antibody, Cell Signaling Technology, 9733S; H3K9me3 antibody, Cell Signaling Technology, 13969) for 2 h, followed by incubation with 50 μL secondary antibody-dig-wash Buffer for 1 h. After washing with dig-wash Buffer, cells were treated with 100 μL Hyperactive pA-Tn5 Transposon (0.04 μM) for 1 h, washed, and resuspended in 300 μL Tagmentation Buffer. After 1 h of incubation at 37°C, the reaction was terminated with 10 μL EDTA, 3 μL SDS, and 2.5 μL Proteinase K, followed by incubation at 50°C for 1 h. DNA was extracted using phenol-chloroform-isoamyl alcohol (25:24:1) and dissolved in 25 μL TE buffer. The CUT&Tag library was amplified with 13 PCR cycles and sequenced on a Novogene HiSeq PE150 platform. Two biological replicates were performed for each condition.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Library name: H3K27me3(sgURE)
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| Data processing |
The CUT&Tag data was processed using CUT-RUNTools-2.0 0 (Yu et al., 2021). First, the input FASTQ files underwent read trimming, mapping, and filtering. Trimmed reads were aligned to the reference genome, and only those with high mapping quality, unique alignment, and proper mapping were retained for further analysis. Simultaneously, spike-in controls were aligned to the E. coli genome. The aligned reads were then normalized based on spike-in sequence depth to account for differences in library size across samples. Peak identification was performed using MACS2 (Zhang et al., 2008), calling narrow peaks for histone modifications H3K27ac, H3K4me3, and H3K9me3, and broad peaks for H2AK119ub, H3K27me3, and H3K4me1.
Assembly: hg19
Supplementary files format and content: CUT&Tag output files: bigwig.
Library strategy: CUT&Tag
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| Submission date |
Jan 02, 2025 |
| Last update date |
Jan 03, 2025 |
| Contact name |
Ke Zhao |
| E-mail(s) |
zk8584918@gmail.com
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| Organization name |
Tianjin Medical University
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| Street address |
Qixiangtai Road, No.22
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| City |
Tianjin |
| ZIP/Postal code |
05000 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE285718 |
Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1 [CUT&Tag] |
| GSE286226 |
Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1 |
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| Relations |
| BioSample |
SAMN46054708 |
| SRA |
SRX27231276 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8707262_sgURE-H3K27me3.bowtie2.fragments.normalized.bw |
14.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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