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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 19, 2016 |
| Title |
PAPERCLIP for pEN2, replicate 1 |
| Sample type |
SRA |
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| Source name |
HeLa cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa cells
type: replicate 1
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| Treatment protocol |
pEN2 transfection
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| Extracted molecule |
total RNA |
| Extraction protocol |
Sample preparation, immunoprecipitation, SDS-PAGE and RNA extraction were adapted from standard HITS-CLIP (Moore et al. 2014). Mouse monoclonal anti-PABP (Sigma, P6246) was used for immunoprecipitation.
The sequencing library was constructed using BrdU-CLIP method (Weyn-Vanhentenryck et al. 2014) with modification to improve sensitivity. The library contains a 14-nt degenerate linker sequence at the 5′ end (6-nt random barcode followed by 8-nt sample multiplexing index). The complete protocol is detailed in Supplemental Method.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
Index 3: CGAT
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| Data processing |
The processing of raw reads was performed using the CIMS software package previously described (Moore et al. 2014). In brief, the raw reads were filtered based on quality score.
Filtered reads with the exact sequence were collapsed into one.
The 5′ degenerate linker was removed.
Poly(A) sequence at the 3′ end was then trimmed using CutAdapt (Martin 2011). Only reads that are at least 25-nt in length are retained for mapping to reference genome (hg19 or mm10).
Mapping was performed using Novoalign (http://www.novocraft.com) without trimming. A minimum of 25-nt match to the genome sequence was required and only those reads mapped unambiguously to the genome (single hits) were kept for downstream analysis.
Reads mapping to the same genomic positions without distinct barcodes were further collapsed into a single tag as previously described (Moore et al. 2014). All reads went through the entire process are referred to as “unique reads” and were used for poly(A) site annotation and other downstream analysis.
Unique reads from both replicates were merged. CIMS software package was used to cluster overlapping reads and to determine the read counts (PH) for each cluster. Only clusters that contained more than 2 (for mouse cortex) or 3 (for cultured cells) unique reads were used for downstream analysis. For each filtered cluster, the most abundant 3′ end from all reads in the cluster was assigned as the poly(A) site. The 3′ most position was selected if there was a tie in abundance for multiple 3′ ends. Please refer to the citation for further information.
Genome_build: hg19
Supplementary_files_format_and_content: bed files for uniquely mapped reads
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| Submission date |
Aug 19, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Hun-Way Hwang |
| E-mail(s) |
Hunway.Hwang@pitt.edu
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| Organization name |
University of Pittsburgh
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| Department |
Department of Pathology
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| Lab |
S754 Scaife Hall
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| Street address |
3550 Terrace Street
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15261 |
| Country |
USA |
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| Platform ID |
GPL15520 |
| Series (1) |
| GSE66092 |
PAPERCLIP Identifies MicroRNA Targets and a Role of CstF64/64tau in Promoting Non-canonical poly(A) Site Usage. |
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| Relations |
| BioSample |
SAMN04001680 |
| SRA |
SRX1159879 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1857621_B5p134_No3.WT.trim.tag.uniq.bed.txt.gz |
16.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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