|
| Status |
Public on May 11, 2020 |
| Title |
MED14_dTAG_7_A |
| Sample type |
SRA |
| |
|
| Source name |
KBM7_MED14-dTAG_6h 500nM dTAG7
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: KBM7
cell type: chronic myeloid leukemia cell line
genotype: MED14-dTAG
treatment: 6h 500nM dTAG7
|
| Treatment protocol |
Cells were treated for 6h with 500nM dTAG7, 100nM dBET6, 250nM THAL-SNS-032 (dCDK9) from 1000x stocks or DMSO. Cells were quantitatively harvested by centrifugation, washed with PBS and snap-frozen in liquid nitrogen.
|
| Growth protocol |
KBM7 cells were grown in IMDM medium supplemented with 10% FBS and 1% Pen/Strep. 48h prior to treatment, all cell lines were seeded to 500k cells/mL in 10mL to T25 flasks. For treatment, 800k cells/condition were seeded in 1mL to 24-well plates in triplicates.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated using commercially available kits (RNeasy mini, Qiagen). For cell-count normalization, equal amounts (10µL of 1:100 dilution = 0.303ng) of synthetic mRNA spike-ins (SIRV-Set-3; Lexogen, Vienna, Austria) were added to QIAshredder-homogenized lysates after the first extraction step with buffer RLT. Total RNA was quantified using NanoDrop and diluted to 70ng/uL for library preparation.
Quant-seq libraries were generated from 350ng RNA using a commercially available kit (Lexogen, Vienna, Austria). Samples were PCR-amplified for 13 cycles, yielding 0.67-3.16ng/µL DNA per sample. Samples were pooled to equimolar amounts for 50bp sequencing in single-read mode.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Data processing |
conversion and demultiplexing: Illumina2bam
FASTQ conversion: bamtools v2.3.0 convert -format fastq
trimming: BBMAP v38.00 'bbduk.sh -ref "polyA.fa.gz","truseq.fa.gz" k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20'
alignment: STAR v2.5.2b to a concatenated hg38_SIRV-Set-3 index with '--outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.6 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outSAMattributes NH HI NM MD --outSAMtype BAM SortedByCoordinate'
read counting: 'htseq-count v0.6.0 -m intersection-nonempty -s yes -f bam -r pos' on a hg38_SIRV-Set-3.gtf annotation
normalization: LOESS normalization was performed based on SIRV-Set-3 spike-ins using R library affy v1.50.0 normalize.loess
Genome_build: hg38
Supplementary_files_format_and_content: bigWig generation: strand-specific signal tracks were computed with 'bedtools v2.26.0 genomeCoverageBed -bg -strand' followed by UCSC 'bedGraphToBigWig v4'
|
| |
|
| Submission date |
Oct 28, 2019 |
| Last update date |
May 12, 2020 |
| Contact name |
Georg E Winter |
| Organization name |
CeMM - Research Institute for Molecular Medicine of the Austrian Academy of Sciences
|
| Street address |
Lazarettgasse 14, AKH BT25.3
|
| City |
Wien |
| State/province |
Austria |
| ZIP/Postal code |
1090 |
| Country |
Austria |
| |
|
| Platform ID |
GPL21290 |
| Series (2) |
| GSE139461 |
Selective Mediator-dependence of cell type-specifying transcription [Quant-Seq] |
| GSE139468 |
Selective Mediator dependence of cell-type-specifying transcription |
|
| Relations |
| BioSample |
SAMN13139858 |
| SRA |
SRX7064196 |
