|
| Status |
Public on Jan 21, 2019 |
| Title |
RBFOX2_SpyCLIP |
| Sample type |
SRA |
| |
|
| Source name |
embryonic kidney cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Lenti-X 293T
treatment: none
|
| Treatment protocol |
To induce acute translation suppression, Lenti-X 293T cells were treated with 2μg/ml harrington for 1 hour at 37C before harvesting.
|
| Growth protocol |
Lenti-X 293T cells were maintained in Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum. To produce the lentiviruses, Lenti-X 293T cells were transfected with a virus vector encoding the expression cassette as well as the VSVG and ΔR8.91 plasmids. Viruses were harvested 48 hours post-transfection. RBP-expressing Lenti-X 293T stable cell lines were generated by transduction with lentiviruses in the presence of 8 μg/ml of polybrene overnight, followed by selection with 1 μg/ml puromycin for one week. To induce RBP expression, 1 ng/ml dox was added to the cell culture medium, and the cells were harvested 48 hours after induction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAs were extracted using TRIzol LS reagent, according to the manufacturer’s instructions.
Briefly, UV-crosslinked cells were lysed under mild condition, and RBP-bound RNAs were partially digested with RNase I. The tagged RBP was enriched from the cell lysate by FLAG IP, and a 3’-biotinylated DNA adapter was ligated to the 3’ end of the captured RNA fragments. The ligated RNPs were released into solution by PreScission Protease cleavage and then covalently coupled to the SpyCatcher beads. The covalently linked RNP were extensively washed under denaturing conditions to remove any residual proteins and RNAs bound nonspecifically. The RNA fragments linked to biotinylated 3’ adaptors were released from RBP by proteinase K digestion and captured by Streptavidin beads. After reverse transcription (RT), the cDNAs were ligated to a second DNA adapter containing a unique molecular identifier (UMI-adapter), and the resultant cDNA library was PCR amplified and subjected to deep sequencing.
SpyCLIP sequence
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
RBFOX2 binding RNA
|
| Data processing |
The unique molecular identifier (UMI, 10 nt random sequence) added to the 5' end of each read during library construction was removed and used as the read name in the following analysis.
The adapter sequence at the 3' end of the sequenced reads was removed with the FASTX program (version 0.0.14) (-l 1 -c). Insert cDNAs that were either too short (less than 30 nt) or too long (more than 65 nt) were discarded
The remaining reads were mapped to the human genome (version hg38) using the STAR program with --outFilterMultimapScoreRange 1 --outFilterScoreMin 10 --outFilterMultimapNmax 1 (version 2.5.2b)
Reads that mapped to the same genomic position and had identical UMIs were categorized as PCR duplicates and then collapsed into a single read
Because the 5' end of the insert cDNA was the RBP crosslinking site, peaks were called based on the 5' start position of usable reads using the iCount program with 3 nt clustering
The number of usable reads in each cluster was normalized to RPM (reads per million genome mapped reads) and considered the abundance
To remove noise signals, SpyCLIP clusters were normalized against input clusters. The abundance of each cluster in SpyCLIP and input sample was calculated. SpyCLIP clusters exhibiting at least an 15-fold enrichment over their corresponding input counterparts were regarded as RBP-specific clusters
Genome_build: hg38.p7
Supplementary_files_format_and_content: bed
|
| |
|
| Submission date |
May 21, 2018 |
| Last update date |
Jan 21, 2019 |
| Contact name |
Ligang Wu |
| E-mail(s) |
lgwu@sibcb.ac.cn
|
| Phone |
+86-21-54921321
|
| Organization name |
Chinese Academy of Sciences
|
| Department |
Shanghai Institutes for biological Sciences
|
| Lab |
Wu ligang
|
| Street address |
320 Yueyang Road
|
| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200031 |
| Country |
China |
| |
|
| Platform ID |
GPL20795 |
| Series (1) |
| GSE114720 |
SpyCLIP: An easy-to-use and high-throughput compatible platform for efficient characterization of protein-RNA interactions |
|
| Relations |
| BioSample |
SAMN09232447 |
| SRA |
SRX4107955 |
