|
| Status |
Public on Jun 20, 2017 |
| Title |
nascent RNA-Seq shcontrol replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
Kasumi-1 cells
|
| Organism |
Homo sapiens |
| Characteristics |
shRNA: control shRNA
|
| Treatment protocol |
Newly transcribed RNA (nascent RNA) was labeled by direct administration of 4-thiouridine (Sigma) to cell culture medium to a final concentration of 100 µM for 1 hour.
|
| Growth protocol |
Kasumi-1 cells stably expressing control shRNA or shRNA targeting AES were cultured in RPMI-1640 medium with 20% FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by RNeasy spin column (Qiagen) purification according to manufacturer’s instructions. 100 µg of total RNA were biotinylated with 200 µg/ml EZ-Link HPDP Biotin (Thermo Scientific) in 1 ml labeling buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA). Biotinylated RNA was bound to streptavidin magnetic beads (Thermo Scientific), washed as recommended by the manufacturer and separated by applying RNA-beads to µMACS columns and magnetic stand (Miltenyi Biotec). The retained material was eluted with 100mM DTT. After a final purification on RNeasy columns nascent RNA was subjected to library preparation and next generation sequencing.
Paired-end library was made from the isolated nascent RNA with TruSeq Stranded Total RNA Sample Prep Kit (Illumina) according to the instructions of manufacturer with the exclusion of poly-A purification step.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
The resulting reads were trimmed for adaptor sequence, mapped against the hg19 reference using the aligner STAR with annotation (transcriptome annotation) from the gencode project (version 18).
Cufflinks/Cuffdiff was used to detect differentially transcribed transcripts.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample
|
| |
|
| Submission date |
Apr 22, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Christian Rohde |
| E-mail(s) |
[email protected]
|
| Organization name |
Heidelberg University
|
| Lab |
Molecular Hematology and Oncology
|
| Street address |
Im Neuenheimer Feld 410
|
| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE80582 |
Nascent RNA-seq in Kasumi-1 cells |
| GSE80602 |
AML1-ETO induces leukemia via C/D box snoRNA/RNPs. |
|
| Relations |
| BioSample |
SAMN04886225 |
| SRA |
SRX1720748 |
