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Sample GSM1881512 Query DataSets for GSM1881512
Status Public on Nov 15, 2015
Title Huh-7.5 CLEAR-CLIP9
Sample type SRA
 
Source name Huh-7.5 hepatoma cells
Organism Homo sapiens
Characteristics cell line: Huh-7.5
Treatment protocol LNA-122, miravirsen or siRNA treatment, or HSPC117 over-expression according to protocol described in accompanying publication.
Growth protocol Huh-7.5 cell culture
Extracted molecule total RNA
Extraction protocol Huh-7.5 cells were UV-irradiated. AGO-cross-linked RNA was isolated with the Std. AGO-CLIP or CLEAR-CLIP protocol described in detail in the accompanying publication.
AGO-cross-linked RNA was cloned by addition of pre-adenylated 3' linker and RNA 5' linker, then RT-PCR, as described in the published CLEAR-CLIP protocol. Products were amplified in a second round of PCR that added a 4 nt index (for multiplexing, see above) and 16 nt common primer sequence. Raw files are demultiplexed on the basis of this index, but have not been further modified.
 
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2500
 
Description protocol: CLEAR-CLIP
complex MW (kD): 130-180
multiplex index: AACC
RNAse A (high), HSPC117 over-expression no T4 RNA ligase
Data processing base calling: Casava 1.8.2
quality filtering: minimum quality filter applied for barcode and insert: fastq_filter.pl; -if sanger -f min:0-3:20,mean:20-40:20 -maxN -1 -v -of fasta ; http://sourceforge.net/projects/ngs-cims/files/CIMS.v1.0.5.tgz/download
collapse exact sequences: exact duplicate sequences removed: fasta2collapse.pl; ; http://sourceforge.net/projects/ngs-cims/files/CIMS.v1.0.5.tgz/download
remove 3' adapter sequene: 3' adapter GTGTCAGTCACTTCCAGCGG clipped: fastx Clipper; -l 20 -a GTGTCAGTCACTTCCAGCGG -n; http://hannonlab.cshl.edu/fastx_toolkit/
trim 5' adapter sequence: 5' 20 nucleotides trimmed (NNNNAGGGAGGACGATGCGG, where NNNN is 4 nucleotide index for multiplexing): fastx Trimmer; -f 21; http://hannonlab.cshl.edu/fastx_toolkit/
strip 5' degenerate linker: 5 nucleotide (NNNNG) degenerate barcode stripped and appended to read IDs: stripBarcode.pl; -linker 5 ; http://sourceforge.net/projects/ngs-cims/files/CIMS.v1.0.5.tgz/download
alignment against micoRNA database: microRNA database mapped against sample file (treated as reference genome) to identify potential chimeric reads containing microRNA sequence: Bowtie; -n 1 -l 8 -e 35; http://bowtie-bio.sourceforge.net/index.shtml
extract chimeric read: extract potential chimeric sequence 5' or 3' of mapped microRNA sequence: text manipulation in R; ;
alignment against genome reference: alignment ofpotential chimeric reads against reference genome to identify target sites: Bowtie; -n 1 -l 8 -e 35; http://bowtie-bio.sourceforge.net/index.shtml
collapse PCR duplicates: mapped reads with same 5' end and same degenerate barcode consolidated: tag2collapse.pl; keep-tag-name --keep-max-score --random-linker -EM 30 --seq-error-model em-local --weight --weight-in-name ; http://sourceforge.net/projects/ngs-cims/files/CIMS.v1.0.5.tgz/download
clustering : chimera interactions with the same ligated microRNA and overlapping genomic coordinates were clustered: Genome_Intervals R package; ;
Genome_build: hg18
Supplementary_files_format_and_content: Excel spreadsheet with gene coordinate information and additional annotations for chimera-identified interactions
 
Submission date Sep 15, 2015
Last update date May 15, 2019
Contact name Michael John Moore
Organization name The Rockefeller University
Department Molecular Neuro-Oncology
Lab Robert Darnell
Street address 1230 York Ave Box 226
City New York
State/province NY
ZIP/Postal code 10065
Country USA
 
Platform ID GPL16791
Series (2)
GSE73057 miRNA-target chimeras identified with CLEAR-CLIP reveal miRNA 3' end pairing as a major determinant of Argonaute binding in vivo [human Huh7.5 AGO-CLIP & CLEAR-CLIP]
GSE73059 miRNA-target chimeras identified with CLEAR-CLIP reveal miRNA 3' end pairing as a major determinant of Argonaute binding in vivo
Relations
BioSample SAMN04088079
SRA SRX1247136

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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