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| Status |
Public on Apr 17, 2023 |
| Title |
SUM159 pLV-KRAB + empty vector control, H3K9me3, abcam Ab8898, biol rep 1 |
| Sample type |
SRA |
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| Source name |
SUM159
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| Organism |
Homo sapiens |
| Characteristics |
cell line: SUM159
cell type: Triple-negative breast cancer
transduction (vector): pLV-KRAB
transduction (grna): No targeting gRNA (aka Empty Vector Control; EVC)
chip antibody: H3K9me3 (ab8898)
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| Growth protocol |
F12 Medium supplemented with 0.6% 1M Hepes, 5 µg/mL insulin, 1 µg/mL hydrocortisone, 1% Anti-Anti and 10% heat inactivated FBS
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Changes in histone modification were assessed using a modified Native ChIP protocol adapted from Grzybowski et al, 2019 [10.1038/s41596-019-0218-7] with minor changes from the original protocol as follows. Native ChIPs were performed using Anti-Rabbit IgG isotype (negative control, CST #66362), Anti-H3K4me3 (Abcam ab8580), and Anti-H3K9me3 (Abcam ab8898) in SUM159 All gRNA and No gRNA. Per 6 ChIP reactions, 6 x 106 cells were harvested for nuclei preparation. The M220 Focused-ultrasonicator™ (Covaris) was used to sonicate the chromatin for 3 min at 20% duty cycle, 75W, 200 cycles per burst before proceeding to MNase digestion and HAP purification as per original protocol. Fifteen µL of HAP purified chromatin was set aside as input control.
Following incubation in a C1000 Touch Thermal Cycler (Bio-Rad) at 75 °C with a hot lid ≥ 85 °C for 6 min, final clean-up was performed using 1.5x AMpure beads and purified fragment size and quality were determined using High Sensitivity dsDNA Qubit™ and Agilent Tapestation. DNA from ChIP input and pulldown were subjected to ThruPLEX® DNA-Seq (Takara R400675) library preparation as per manufacturer’s instructions.
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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 |
Basecalls were performed using bcl2fastq v2.17 for Novaseq output.
ChIP-seq reads were trimmed from 3' end until the final base had a quality score > 30, using Trimmomatic v0.33, discarding reads left with < 20 bp
ChIP-seq reads were aligned to the UCSC hg38 genome using Bowtie version 1.1.2. Only uniquely mapping reads with at most two mismatches were retained.
To create ChIP-seq coverage plots, the locations of the mapped ChIP-seq reads were extended to 150 bp to represent sequenced fragments, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.
Peaks were called uing MACS v2.1.0 with the significance cut-off q-value <=0.01
bigWig files were generated using the genomicRanges R package. Score represents the normalized coverage of DNA fragments at a given genomic coordinate. narrowPeak files were generated using MACS v2 with default settings.
Assembly: hg38
Supplementary files format and content: bigWig, narrowPeak (except for Input sample)
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| Submission date |
Aug 01, 2022 |
| Last update date |
Apr 18, 2023 |
| Contact name |
Joe Cursons |
| Organization name |
Monash University
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| Department |
Biomedicine Discovery Institute
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| Street address |
19 Innovation Walk
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| City |
Clayton |
| State/province |
VIC |
| ZIP/Postal code |
3800 |
| Country |
Australia |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE210275 |
Synthetic epigenetic reprogramming of mesenchymal to epithelial states using the CRISPR/dCas9 platform in triple negative breast cancer [ChIP-seq] |
| GSE210277 |
Synthetic epigenetic reprogramming of mesenchymal to epithelial states using the CRISPR/dCas9 platform in triple negative breast cancer |
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| Relations |
| BioSample |
SAMN30088396 |
| SRA |
SRX16761097 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6427516_RL2891_16_empty_vector_H3K09me3_rep1_peaks.broadPeak.gz |
9.2 Mb |
(ftp)(http) |
BROADPEAK |
| GSM6427516_RL2891_2021_12_15_16_empty_vector_H3K09me3_rep1_CPM.bw |
263.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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