|
| Status |
Public on Jan 17, 2013 |
| Title |
Hela_AGO2_CLIP_ptb_knockdown |
| Sample type |
SRA |
| |
|
| Source name |
Hela cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Hela
clip antibody: Anti-AGO2 mAb, clone 2E12-1C9(Abnova)
|
| Treatment protocol |
For CLIP-seq samples, Hela cells were UV-irradiated at 400mj. For pair-end RNA-seq, Hela cells were treated with control shRNA or PTB specific shRNA. For 3' end RNA-seq, Hela cells were further treated with actinomycin D (2 μg/ml) for 4 hours.
|
| Growth protocol |
HeLa cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS (Omega Scientific) and 100U of penicillin/streptomycin (Life Technology).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
CLIP-seq library was prepared according to previously published standard protocol. RNA-seq was typically done after shRNA treatment for 72 hrs. Trizol-isolated RNA was enriched in two rounds for Poly(A+) RNA with paramagnetic oligo(dT), fragmented into ~200 nt in length, converted to cDNA with Superscript III (Invitrogen), and subjected to deep sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
CLIP-seq: After removing 3' adaptor sequence, the tags longer than 20 nt were mapped to the reference human genome (hg18) with Bowtie 0.12.7 by allowing maximum 2 mismatches in the first 25 nt, and only tags with less than or equal to 10 mapped locations were kept.
RNA-seq: Paired-end reads were separately mapped into a custom Bowtie index, which was built from UCSC knownGenes (hg18) not including introns. Under the constraint of the insert size, only the paired-end reads whose two ends are about 50 nt apart on the same gene were kept. For each pair, the coordinates of the two end reads were mapped back to the genome based on its gene's genomic location, and then joined together. Only the pair-end reads with one unique genomic location were kept.
3' end RNA-seq: After removing 3' adaptor sequence, the tags longer than 20 nt were mapped to the reference human genome (hg18) with Bowtie 0.12.7 by allowing maximum 2 mismatches in the first 25 nt, and only tags with less than or equal to 10 mapped locations were kept. Then scanned the genome for polyA-stretch defined as consecutive 8As, removed them; adaptively clustered tags within a specific distance 30 nt along transcripts and sorted cluster intervals by the length and tag in a decreasing order. If a cluster contains a known 3’end within a 300 nt window, we used the end and then counted the number of reads in each cluster.
Genome_build: UCSC hg18
Supplementary_files_format_and_content: bed format
|
| |
|
| Submission date |
Dec 03, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Jie Huang |
| E-mail(s) |
jih052@ucsd.edu
|
| Phone |
8588882373
|
| Fax |
858-822-6920
|
| Organization name |
UC San Diego
|
| Department |
Department of Cellular and Molecular Medicine
|
| Lab |
George Palade Laboratories
|
| Street address |
9500 Gilman Drive, Room 231
|
| City |
La Jolla, San Diego |
| State/province |
CA |
| ZIP/Postal code |
92093-0651 |
| Country |
USA |
| |
|
| Platform ID |
GPL9115 |
| Series (1) |
| GSE42701 |
Direct Conversion of Fibroblasts to Neurons by Reprogramming PTB-Regulated microRNA Circuits |
|
| Relations |
| SRA |
SRX208352 |
| BioSample |
SAMN01822953 |
