|
| Status |
Public on Feb 27, 2019 |
| Title |
ALYREF-iCLIP SLBP KD-2 |
| Sample type |
SRA |
| |
|
| Source name |
ALYREF-iCLIP SLBP KD
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
sirna treatment: SLBP knockdown
antbody: ALYREF antibody (ABclonal, A17974)
|
| Treatment protocol |
The ALYREF-iCLIP assay was performed in control and SLBP knock down cells using ALYREF antibody. The NXF1-iCLIP experiments were carried out in Flag-NXF1 stable expressed cells treated with control, ALYREF and SLBP siRNAs for 72 hr using Flag antibody.
|
| Growth protocol |
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Biochrom)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Briefly, the cells for ALYREF iCLIP or NXF1-iCLIP were irradiated with UV light at 200 mJ/cm2 or 400 mJ/cm2. After cell lysis, RNAs were partially fragmented using RNase I, followed by IPs with the ALYREF antibody immobilized on protein A Dynabeads (Life Technologies) or the Flag antibody immobilized on protein G Dynabeads (Life Technologies). After extensive washing, immunoprecipitated RNAs were ligated at the 3’ ends to a RNA adapter and radioactively labeled by T4 polynucleotide kinase (Fermentas). The protein-RNA complexes were then transferred to a nitrocellulose membrane. Then, fragmented RNAs were purified from nitrocellulose membrane by PK treatment and phenol / chloroform extranction.
For iCLIP cDNA library preparation, fragmented RNAs, which were ligated with a RNA adapter (3' linker) at the 3’ ends , reverse-transcribed with a primer containing a barcode (iCLIP RT primer). The resulting cDNAs were purified by PAGE, circularized by single-stranded DNA ligase (Epicentre), linearized by restriction enzyme cleavage, and amplified and add P5/P3 adaptors by PCRs with (P3 and P5 solexa primer).
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
library strategy: iCLIP-seq
Briefly, custom scripts were used to demultiplex the different experimental samples by their sample barcodes. The reads containing adaptors were trimmed with cutadapt program and reads less than 18 bp were discard.
The remaining reads were then mapped to the human genome (hg19) including splicing junctions from GENCODE gene annotation (v19) with STAR. Usable reads obtained by removing potential PCR duplicates from mapped reads were used for further downstream analysis
Genome_build: hg19
Supplementary_files_format_and_content: bed
|
| |
|
| Submission date |
Jan 07, 2019 |
| Last update date |
Feb 27, 2019 |
| Contact name |
Hong Cheng |
| E-mail(s) |
hcheng@sibcb.ac.cn
|
| Organization name |
Shanghai Institutes for Biological Sciences
|
| Department |
Shanghai Institute of Biochemistry and Cell Biology
|
| Lab |
StateKey Laboratory of Molecular Biology
|
| Street address |
320 YueYang Road
|
| City |
Shanghai |
| ZIP/Postal code |
200031 |
| Country |
China |
| |
|
| Platform ID |
GPL20795 |
| Series (2) |
| GSE124735 |
ALYREF is shared by polyadenylated and nonpolyadenylated mRNA processing and export pathways [iCLIP-seq] |
| GSE125005 |
ALYREF is shared by polyadenylated and nonpolyadenylated mRNA processing and export pathways |
|
| Relations |
| BioSample |
SAMN10700552 |
| SRA |
SRX5213356 |
