|
| Status |
Public on Dec 15, 2016 |
| Title |
Lib153OverExpressTRUB2rep2_input |
| Sample type |
SRA |
| |
|
| Source name |
OverExpressTRUB2
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: input
cell line: HEK293
|
| Treatment protocol |
siRNAs targeting TRUB1, TRUB2, and PUS7 (catalog numbers: s4111 and s4112) were transfected using Lipofectamine RNAiMAX (Life Technologies) following the manufacturer’s protocols, with two siRNA boosts at 48 and 96 hours following transfection; As negative controls, we used Ambion® In Vivo Negative Control #1 siRNA (catalog number: 4457287). Cells were harvested at 144 hours. For overexpression, plasmids encoding full length TRUB1 and TRUB2 were obtained from DNASU (HsCD00039729 and HsCD00041101, respectively) and cloned into a Gateway destination vector, downstream of an EF1ɑpromoter. The plasmids were transfected into HEK293 cell using either Lipofectamine LTX reagent (Life technologies) or polyJet (signagen) with one boost of the plasmid at 24 hours. Cells were harvested 48 hours following transfection.
|
| Growth protocol |
Human HEK293 cells were plated in 6-well plates at 20% confluence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Enrichment of polyadenylated RNA (polyA+ RNA) from total RNA was performed using one round of enrichment using Oligo(dT) dynabeads (Invitrogen) according to the manufacturer’s protocol. CMC-treatment was performed essentially as described in (Schwartz et al, Cell, 2014).
The mRNA was chemically fragmented into ~80-150 nt-long fragments by performing a short fragmentation (30 seconds) using RNA fragmentation reagent and stop solution (Ambion). RNA was subjected to FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), followed by a 3’ ligation of an RNA adapter using T4 ligase (New England Biolabs). Ligated RNA was reverse transcribed using AffinityScript Multiple Temperature Reverse Transcriptase (Agilent) or SuperScript III (Life Technologies), and the cDNA was subjected to a 3’ ligation with a second adapter using T4 ligase. The single-stranded cDNA product was then amplified for 9-12 cycles in a PCR reaction. Libraries were sequenced on Illumina HiSeq 2500 platforms generating paired end reads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Reads were aligned to the human genome (hg19) using STAR aligner with default parameters
An in house script was used to merge left and right reads into a single bam file spanning the full interval from the beginning of the 'left' read to the end of the 'right' read
Strand specific bedgraph files were generated quantifying the number of reads beginning at each position, using bedtools genomecov (activating the '-5' and '-strand' flags
Genome_build: hg19
Supplementary_files_format_and_content: bedgraph
|
| |
|
| Submission date |
Dec 04, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Schraga Schwartz |
| Organization name |
WEIZMANN INSTITUTE OF SCIENCE
|
| Street address |
Herzl 234, Department of Molecular Genetics
|
| City |
Rehovot |
| State/province |
Choose a State or Province |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE90851 |
Predominant TRUB1-dependent pseudouridylation of mammalian mRNA via a predictable and conserved code |
|
| Relations |
| BioSample |
SAMN06108419 |
| SRA |
SRX2394558 |
