|
| Status |
Public on Jul 28, 2013 |
| Title |
RNA-Seq USP49 knockdown |
| Sample type |
SRA |
| |
|
| Source name |
colorectal carcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
treatment: USP49 knockdown
|
| Treatment protocol |
USP49 knockdown was induced by adding doxycycline to growth media. Cells were harvested 36 hours after doxycycline induction.
|
| Growth protocol |
HCT116 USP49 tetracycline inducible knockdown cells (T-Rex Expression System) were grown in DMEM supplemented with 10% FBS, 1% Pennicillin and Streptomycine, blasticidin and puromycin.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
For RNA-Seq, mRNA was specifically enriched from total RNA using oligo(dT) beads and sheared into small pieces. The fragments were then reverse-transcribed into first-strand cDNA using random hexamer primers, followed by second strand synthesis using DNA polymerase I. The short cDNA strands were ligated with 3' and 5' adapters for amplification and sequencing. For ChIP-seq, uninduced control and induced knockdown cells were crosslinked with 1% formaldehyde and chromatin was disrupted into 100-400 bp fragments by sonication and precipitated with either anti-uH2B or anti-H2B antibodies. After elution from the beads and reverse cross-linking, the DNA fragments were extracted with phenol/chloroform and recovered by ethanol precipitation. Immunoprecipitated DNA was ligated with 3' and 5' adapters for amplification and sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1000 |
| |
|
| Data processing |
Basecalls for both RNA-Seq and ChIP-Seq were performed in Illumina 1.5 software.
RNA-Seq and ChIP-seq files were were mapped to hg18 using TopHat (RNA-seq) or Bowtie (ChIP-seq). Samtools and Picard were used for post alignment processing.
FPKM and transcript sizes were determined using Cufflinks with default settings.
Genome_build: hg18
Supplementary_files_format_and_content: Mapped reads were converted to bedgraph format (BEDTools) and normalized as reads per million total unique mapped reads using the -scale parameter. GTF files for control assembled transcripts and Usp49 knockdown assembled transcripts.
|
| |
|
| Submission date |
Jun 17, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Hengbin Wang |
| Organization name |
Virginia Commonwealth University
|
| Department |
Massey Cancer Institute
|
| Street address |
401 College Street
|
| City |
Richmond |
| State/province |
VA |
| ZIP/Postal code |
23298 |
| Country |
USA |
| |
|
| Platform ID |
GPL15433 |
| Series (2) |
| GSE38100 |
Examination of gene expression in wild type and USP49 knockdown cells [RNA-Seq] |
| GSE38101 |
USP49 deubiquitinates histone H2B and regulates pre-mRNA splicing |
|
| Relations |
| BioSample |
SAMN02205197 |
| SRA |
SRX306929 |
