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Sample GSM1857616 Query DataSets for GSM1857616
Status Public on Feb 19, 2016
Title PAPERCLIP for si-Ctrl in LN229, replicate 2
Sample type SRA
 
Source name LN229 cells
Organism Homo sapiens
Characteristics cell line: LN229 cells
type: replicate 2
Treatment protocol si-Ctrl
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.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina MiSeq
 
Description Index 6: GACT
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
 
Submission date Aug 19, 2015
Last update date May 15, 2019
Contact name Hun-Way Hwang
E-mail(s) Hunway.Hwang@pitt.edu
Organization name University of Pittsburgh
Department Department of Pathology
Lab S754 Scaife Hall
Street address 3550 Terrace Street
City Pittsburgh
State/province PA
ZIP/Postal code 15261
Country USA
 
Platform ID GPL15520
Series (1)
GSE66092 PAPERCLIP Identifies MicroRNA Targets and a Role of CstF64/64tau in Promoting Non-canonical poly(A) Site Usage.
Relations
BioSample SAMN04001675
SRA SRX1159874

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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