|
| Status |
Public on Nov 18, 2014 |
| Title |
PCBP2 iCLIP in JFH-1 Huh7 |
| Sample type |
SRA |
| |
|
| Source name |
HUH7
|
| Organism |
Homo sapiens |
| Characteristics |
host cell line: HUH7
infection: JFH-1 isolate of HCV
rip antibody: anti-PCBP2 (Sigma Aldrich, catalog number R4155)
|
| Treatment protocol |
Huh7 cells were either uninfected or infected with the JFH-1 isolate of HCV at a multiplicity of infection of 0.01
|
| Growth protocol |
Huh7 cells were grown in DMEM with 10% FBS, 1% nonessential amino acids, and 200uM L-glutamine
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lysates were clarified from sonicated Huh7 cells and PCBP2-RNA complexes were isolated with an anti-PCBP2 antibody.
Libraries were prepared according to the standard iCLIP protocol (PMID: 24184352), substituting the 3' RNA adaptor for one with a 3'-Biotin blocking moity. Subsequent steps utalized a Streptavidin-Biotin capture strategy to eliminate the need for precipiations between biochemical reactions. Each sequencing read conatins a 13 nt random (D's and N's) and experimental (XXXX) "DDDNNXXXXNNNN". The experimental barcodes are described in (PMID: 24184352)
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
replicates 1 and 2
library strategy: iCLIPseq
|
| Data processing |
Reads were filtered for quality, PCR duplicates removed using the random barcode, 5' and 3' adaptor sequences were trimmed and reads were mapped to hg19 and a custom noncoding RNA index [We constructed a custom index of human short repetitive noncoding RNAs (including snRNAs and Y RNAs) as well as the full 42kb human rDNA locus. These sequences were obtained from Rfam (for short repetitive RNAs) and GenBank (for the rDNA locus, GenBank ID: U13369.1). The index was build with each RNA as a tandem element in a single FASTA file which was then used to make a bowtie2 index. This bowtie2 index was then used to map sequencing reads after which unmapped reads were aligned to the hg19 build of the human genome.].
Crosslinked nucleotides were identified as the first nucleotide of each trimmed read. Crosslinked nucleotides were only analyzed if present at least 6 between the biological replicates.
Genome_build: hg19; AB047639.1
Supplementary_files_format_and_content: The bigWig file provided contains processed Crosslinked nucleotides from the DDX21_SAT iCLIP experiment (i.e., trimmed, mapped, and processed data mapping to hg19, excluding reads that mapped to the custom index we built). Data presented is an intersection of the two biological replicates.
|
| |
|
| Submission date |
Jul 28, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ryan Alexander Flynn |
| E-mail(s) |
raflynn@stanford.edu
|
| Organization name |
Stanford University
|
| Street address |
380 Roth Way, Room 265
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE59840 |
Dissecting non-coding and pathogen RNA-protein interactomes |
|
| Relations |
| BioSample |
SAMN02943198 |
| SRA |
SRX663480 |
