|
| Status |
Public on May 09, 2022 |
| Title |
G401_shNR6_GSK126_H3K27ac |
| Sample type |
SRA |
| |
|
| Source name |
G401
|
| Organism |
Homo sapiens |
| Characteristics |
genotype: G401 loss NSD1
agent: EZH2i
|
| Treatment protocol |
G401 cells were engineered to express either GFP or SMARCB1 (B1) under doxycycline treatment and further transfected with either control (sgC) or NSD1 (sgN) targeting sgRNAs. Cells were then treated with 1ug/mL dox for 24h and collected for RNA extraction. For EZH2 inhibition studies, G401 cells stably expressing shCTRL or shNSD1 RNAs were treated for 72h with 1uM GSK126 and collected for RNA extraction.
|
| Growth protocol |
G401 cells were grown in McCoy's 5A media supplemented with 10% fetal bovine serum and 1% Glutamax (Gibco) at 37°C and 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
In brief, cells were cross-linked directly on the plate using 1% paraformaldehyde for 10 min at room temperature and quenched with glycine. Nuclei were isolated using the Covaris TruCHIP kit and Chromatin extracts were sonicated for 8 min using a Covaris E220 focused ultrasonicator at a peak power of 140, a duty factor of 5 and 200 cycles per burst. Chromatin immunoprecipitation was carried overnight with antibodies bound to dynabeads. Chromatin was de-crosslinked, digested with RNase and Proteinase K and DNA was ourified using a Zymo ChIP DNA kit.
KAPA HyperPrep Kit (Kapa Biosystems)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
G401 loss NSD1 EZH2i treated
|
| Data processing |
trim_galore v0.6.3 remove adapter sequences
align quality reads using bwa mem v0.7.17 to a hybrid reference genome
bamsormadup v2.0.87
samtools used to split bam file for hg19 and dm6 reads base on chromosome names
samtools -F 1024 -q 1 to define quality reads
estimate fragment size using spp R package
slopBed -r fragment size estimation determined by spp
Normalize to 15M reads (for canonical normalization) or 100k dm6 reads using bedtools genomecov and conert to bigwig using UCSC bedGraphToBigWig or spikeinfree normalization (https://github.com/stjude/ChIPseqSpikeInFree)
Genome_build: hg19+dm6 (Gencode GRCh37 and Ensembl r97)
Supplementary_files_format_and_content: normalized coverage in the bigwig file format.
Supplementary_files_format_and_content: peak files in bed and narrowPeak format.
|
| |
|
| Submission date |
Jun 18, 2021 |
| Last update date |
Dec 13, 2022 |
| Contact name |
Jacquelyn A Myers |
| E-mail(s) |
[email protected]
|
| Organization name |
St. Jude Children’s Research Hospital
|
| Street address |
262 Danny Thomas Pl
|
| City |
Memphis |
| State/province |
TN |
| ZIP/Postal code |
38105 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE178489 |
Inactivation of the histone H3 K36 methyltransferase NSD1 confers resistance to EZH2 inhibition [ChIP-Seq] |
| GSE178490 |
Inactivation of the histone H3 K36 methyltransferase NSD1 confers resistance to EZH2 inhibition |
|
| Relations |
| BioSample |
SAMN19776586 |
| SRA |
SRX11181489 |
