|
| Status |
Public on May 23, 2021 |
| Title |
S2 Cas9 sgINTS6D CDK9i RNAPII CTD4H8 |
| Sample type |
SRA |
| |
|
| Source name |
Drosophila fruit fly embryonic cell line expressing Cas9 and INTS6 targeting sgRNA
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: Schneiders Drosophila Line 2 (S2)
genotype: expressing Cas9 and INTS6 targeting sgRNA
chip antibody: RNA polymerase II, clone CTD4H8 (Millipore 05-623)
treatment: CDK9i (AZ5576) 100nM 2 hours
|
| Treatment protocol |
Cells were treated with DMSO (untreated control), CDK9i (AZ5576, 100nM or 170nM 2 hours) or DBK-1154 (10µM 2 hours 15 minutes)
|
| Growth protocol |
THP-1 cells were cultured in RPMI-1640 supplemented with 10-20 percent heat-inactivated fetal bovine serum, 100U/mL penicillin, 100µg/mL streptomycin and 2mM GlutaMAX at 37 degrees celciuus and 5 percent carbon dioxide. S2 cells were cultured in Schneider's Drosophila medium supplemented with 10 percent heat-inactivated fetal bovine serum, 100U/mL penicillin, 100µg/mL streptomycin and 2mM GlutaMAX at room temperature and atmospheric carbon dioxide
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked with formaldehyde solution, nuclei were extracted and sonicated using a Covaris. Protein-bound chromatin was isolated using indicated antibody and decrosslinked DNA was isolated using the Zymogen ChIP DNA Clean and Concentrator Kit
ChIP-seq librarys were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB E7645) and 200 - 500 base pair fragments were selected using a Pippin Prep (1.5% agarose, Sage Science)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Sequenced reads were demultiplexed using bcl2fastq (v2.17.1.14)
Basic QC performed on fastq files using FASTQC (v0.11.6)
Fastq files were aligned to a Hg19 or dm3 reference genome performed with Bowtie2 (v2.3.4.1) to generate SAM files
SAM files converted to BAM files using Samtools (v1.9) using view command. BAM files were sorted, indexed and potential PCR duplicates removed using rmdup function
bigWig files were generated from sorted, indexd BAM files using the Deeptools (v3.0.0) BamCoverage function (--normalize using CPM, --smoothLength 150 --binSize 50 -e 200 scaleFactor 1)
Average read density across defined genomic regions were defined using the Deeptools computeMatrix function and plotHeatmap function was used to generate chromatin occupancy heatmaps
Subread (v2.0.0) featureCounts function (-O -M -T 16 -F SAF) was used to count reads within TSS and gene body regions using bedtools (v2.27.1) slop function using UCSC Hg19 or dm3 chromosome sizes
Read density was calculated as RPKM in Rstudio (v3.6.1) using limma (v3.40.6) and edgeR (v.3.26.8) and visualized using ggplot2 (v3.3.1)
Genome_build: Hg19 or dm3
Supplementary_files_format_and_content: bigWig files of RNAPII, CDK9, INTS11, INTS6, BRD4 and RNAPII phospho-ser 2 occupancy across the genome in CPM
|
| |
|
| Submission date |
Dec 24, 2020 |
| Last update date |
May 23, 2021 |
| Contact name |
Stephin J Vervoort |
| E-mail(s) |
[email protected]
|
| Organization name |
Peter MacCallum Cancer Centre
|
| Street address |
305 Grattan Street
|
| City |
Melbourne |
| ZIP/Postal code |
3000 |
| Country |
Australia |
| |
|
| Platform ID |
GPL19132 |
| Series (2) |
| GSE163804 |
A PP2A-Integrator complex fine-tunes transcription by opposing CDK9 [ChIP-seq] |
| GSE163805 |
A PP2A-Integrator complex fine-tunes transcription by opposing CDK9 |
|
| Relations |
| BioSample |
SAMN17152123 |
| SRA |
SRX9730754 |
