|
| Status |
Public on Aug 20, 2015 |
| Title |
hDIS3_WT_RNA-seq |
| Sample type |
SRA |
| |
|
| Source name |
HEK293 Flip-In T-Rex cell line
|
| Organism |
Homo sapiens |
| Characteristics |
variant: DIS3wild type + shRNA
|
| Treatment protocol |
Expression of exogenous genes was induced by addition of doxycycline at a final concentration of 100ng/ml.
|
| Growth protocol |
HEK293 Flp-In T-Rex cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco), supplemented with 10% fetal bovine serum (FBS; Gibco) and penicillin/streptomycin (Sigma-Aldrich) at 37°C in a 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using TRI-Reagent (Sigma-Aldrich)
Total RNA was treated with Turbo-DNAse (Life Technologies), and then rRNA was removed from the samples using Ribo-Zero Kit (Epicentre), according to the manufacturer's protocols. As a control of experiment performance, external RNA (ERCC RNA Spike-In Mix; Life Technologies) was added to the samples. RNA libraries were prepared using TruSeq RNA Sample Preparation Kit (Illumina)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
Basecalls and demultiplexing were performed using CASAVA version 1.8.2
Adapters and reads shorter than 12 nt. and with quality lower than 20 were removed by cutadapt-1.2.1
Reads were mapped using RUMv2.05_05. The program requires left and right reads to be the same length, so reads has been striped adequately. The information about known transcripts was supplied to the program. It contained i) Gencode v18 transcript data ii) supplemented to Gencode v18 data tRNA coordinates from tRNA scan program iii) previously published PROMPT data which is a set 3kB upstream regions from the selected 2471 genes iv) snoRNA precursors defined by us for intronic snoRNA as regions from the end of the snoRNA to the beginning of the exon.
Only uniquely mapped reads have been counted.
New transcripts were assembled from deeply sequenced libraries from PIN RNB double mutant using Cufflinks v 2.1.1. Reads from three replicates were assembled separately. We applied all default parameters with exception of '--max-bundle-length' increased to 10^7 and '--max-bundle-frags' increased to 10^7. We used cuffmerge from cufflinks package to merge assemblies from the replicates.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include raw counts of sequencing reads for each sample replicate; bedgraph files were generated using RUMv2.05_05; gtf file was generated using cuffmerge from Cufflinks v 2.1.1.
|
| |
|
| Submission date |
Dec 18, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrzej Dziembowski |
| E-mail(s) |
andrzejd@ibb.waw.pl
|
| Phone |
+48 22 5922033
|
| Organization name |
Institute of Biochemistry and Biophysics Polish Academy of Sciences
|
| Lab |
Laboratory of RNA Biology and Functional Genomics
|
| Street address |
Pawinskiego 5A
|
| City |
Warszawa |
| ZIP/Postal code |
02-106 |
| Country |
Poland |
| |
|
| Platform ID |
GPL18460 |
| Series (1) |
| GSE64332 |
Human DIS3 shapes the RNA polymerase II transcriptome degrading variety of unwanted transcripts. |
|
| Relations |
| BioSample |
SAMN03268752 |
| SRA |
SRX817371 |
