|
| Status |
Public on May 23, 2021 |
| Title |
THP-1 sgINTS6 CDK9i 6 hours replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
Acute myeloid leukemia cell line expressing Cas9 and INTS6 targeting sgRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: THP-1 cell line
genotype: expressing Cas9 and INTS6 targeting sgRNA
treatment: CDK9i (AZ5576) 170nM for 6 hours, 500µM 4-thiouridine for final 30 minutes
spike-in: Drosophila melanogaster spike-in
|
| Treatment protocol |
THP-1 cells were treated with DMSO for 6 hours (untreated control; CDK9i 0 hours) or CDK9i (AZ5576) 170nM for 2 hours and 6 hours; cells were labelled with 4-thiouridine during the final 30 minutes of CDK9i treatment. S2 cells were labelled with 4-thiouridine for 30 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 |
total RNA |
| Extraction protocol |
Total RNA was isolated by phenol-chloroform extraction and isopropanol precipitation. 4-thiouridine labelled RNA was isolated from total RNA of THP-1 (human) and S2 (Drosophila; 5 percent) cells following biotinylation with EZ-Link HPDP Biotin (ThermoFisher Scientific) and strepavidin µMACs enrichment (Miltenyi). Biotin-enriched 4-thiouridine labelled nascent RNA was isolated using the Qiagen RNAeasy MinElute kit.
RNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEB E7760S)
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
5-T6-2
featureCounts_output_hg19-dm3.txt
201202_4sUseq_SpikeIn_normalisedCounts.csv
|
| Data processing |
library strategy: 4sU-seq
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 combined Hg19/dm3 genome performed with Bowtie2 (v2.3.4.1) to generate SAM filles
SAM files converted to BAM files using Samtools (v1.9) using view command. BAM files were sorted and indexed
For normalization of hg19 reads to Drosophila spike-in, reads mapping to Hg19 and dm3 genomes were calculated using Subread (v2.0.0) featureCounts function. A scale factor was determined by calculating the proportion of reads mapping to the dm3 genome relative to the combined Hg19/dm3 genome
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) using the appropriate scaleFactor
Scaled bigWig files were visualized in IGV and analyzed using the Deeptools (v3.0.0) bigwigCompare function
Data was visualized in Rstudio using ggplot2 (v3.3.1), ggrepel (v3.3.1) and ggfortify (v0.4.10)
Genome_build: Hg19 / dm3
Supplementary_files_format_and_content: bigWig files of nascent RNA coverage in cpm across the genome; text files of featureCounts counts; text file of S2 normalized counts
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| |
|
| 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 |
GPL18573 |
| Series (2) |
| GSE163803 |
A PP2A-Integrator complex fine-tunes transcription by opposing CDK9 [4sU-seq] |
| GSE163805 |
A PP2A-Integrator complex fine-tunes transcription by opposing CDK9 |
|
| Relations |
| BioSample |
SAMN17152105 |
| SRA |
SRX9729927 |
