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Sample GSM545215 Query DataSets for GSM545215
Status Public on May 21, 2010
Title AGO4
Sample type SRA
 
Source name cultured cells, AGO4
Organism Homo sapiens
Characteristics cell line: HEK 293
crosslinker and crosslinking wavelength: 4-SU / 365 nm
antibody: anti-FLAG antibody
antibody vendor: Sigma
antibody catalog #: F1854
immunoprecipitated protein: AGO4
weight of protein recovered: 100 kDa
Treatment protocol For UV crosslinking, cells were washed once with ice-cold PBS while still attached to the plates. PBS was removed completely and cells were irradiated on ice with 365 nm UV light in a Stratalinker 2400 (Stratagene). Cells were scraped off with a rubber policeman in 1 ml PBS per plate and collected by centrifugation at 500×g for 5 min. Detailed experimental procedure can be found in the Supplement to PMID 20371350.
Growth protocol HEK293 T-REx Flp-In cells (Invitrogen) were grown in D-MEM high glucose with 10% (v/v) fetal bovine serum, 1% (v/v) 2 mM L-glutamine, 1% (v/v) 10,000 U/ml penicillin/10,000 µg/ml streptomycin, 100 µg/ml zeocin and 15 µg/ml blasticidin. Cell lines stably expressing FLAG/HA-tagged proteins were generated by co-transfection of pFRT/TO/FLAG/HA or pFRT/FLAG/HA constructs with pOG44 (Invitrogen). Cells were selected by exchanging zeocin with 100 µg/ml hygromycin. Expression of FLAG/HA-IGF2BP1, -2, -3 and TNRC6A, B and C was induced by addition of 250 ng/ml doxycycline 15 to 20 h before crosslinking. Detailed experimental procedure can be found in the Supplement to PMID 20371350.
Extracted molecule total RNA
Extraction protocol The pellets of cells crosslinked with UV 365 nm were resuspended in NP40 lysis buffer and incubated on ice for 10 min. The cell lysate was cleared by centrifugation at 13,000g. RNase T1 (Fermentas) was added to the cleared cell lysates and the reaction mixture was incubated at 22ºC for 15 min and subsequently cooled for 5 min on ice before addition of antibody-conjugated magnetic beads.
The recovered RNA was carried through a cDNA library preparation protocol originally described for cloning of small regulatory RNAs (Hafner et al., 2008). The first step, 3' adapter ligation, was carried out as described on a 20 ul scale using 10.5 ul of the recovered RNA. UV 365 nm crosslinked sample RNAs were processed using Solexa sequencing adapter sets. Depending on the amount of RNA recovered, 5'-adapter-3'-adapter products without inserts may be detected after amplification of the cDNA as additional PCR bands. In such case, the longer PCR product of expected size was excised from a 3% NuSieve low-melting point agarose gel, eluted from the gel pieces with the Illustra GFX-PCR purification kit (GE Healthcare) and Solexa sequenced. Detailed experimental procedure can be found in the Supplement to PMID 20371350.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer
 
Description HEK293 cells stably expressing FLAG/HA-tagged AGO4. Protein was immunoprecipitated by anti-FLAG antibody, Sigma F1854.

Sample: Length of RNA-segment (nt) used for cDNA generation
Ago4_1: 19-24
Ago4_2: 19-24

Combined processed data from AGO4 libraries.
Data processing The basic method for removing adaptors and assigning a functional annotation to the sequence reads was described in (Berninger et al., 2008). Briefly, we used an in-house ends-free local alignment algorithm (score parameters: 2 for match, -3 for mismatch, -2 for gap opening, -3 for gap extension) to align the Solexa adapter to the 3' end of each sequence read, allowing for the possibility that the adapter was not completely sequenced (Software can be downloaded from http://www.mirz.unibas.ch/restricted/clipdata/RESULTS/index.html). We removed from the reads the fragments that aligned to the adaptor as long as the number of matches exceeded that of mismatches by at least 3. Sequences that were either too short (less than 20 nt) or too repetitive (using a cut-off of 0.7 and 1.5 in the entropy of the mono- and dinucleotide distributions, respectively, of individual sequence reads (Berninger et al., 2008)) were discarded because they would probably map to multiple genomic locations. The remaining sequences were mapped to the hg18 version of the human genome assembly that was downloaded from the University of California at Santa Cruz (http://genome.cse.ucsc.edu) and to a database of sequences whose function (rRNA, tRNA, sn/snoRNA, miRNA, mRNA, etc.) is already known. These were obtained from the sources specified in (Berninger et al., 2008). The Oligomap algorithm (Berninger et al., 2008) was used for this purpose, and all the perfect and 1-error (mismatch or insertion or deletion (indel) mappings were obtained. Based on the GMAP (Wu and Watanabe, 2005) genome mapping of human mRNA transcripts from NCBI downloaded on November 4th, 2008, we determined whether the sequence reads mapped to intronic or exonic regions of genes. Based on the coding region annotation of transcripts in GenBank, we determined whether the exonic sequence reads originated from the 5'UTR, CDS or 3'UTR. Detailed bioinformatics procedure can be found in the Supplement to PMID 20371350.
 
Submission date May 19, 2010
Last update date May 15, 2019
Contact name Markus Hafner
E-mail(s) mhafner@rockefeller.edu
Phone +1 212 327 7696
Fax +1212 327 7652
Organization name The Rockefeller University
Department Laboratory of RNA Molecular Biology
Lab Tuschl lab
Street address 1230 York Ave, Box 186
City New York
State/province NY
ZIP/Postal code 10065
Country USA
 
Platform ID GPL9052
Series (2)
GSE21578 Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP
GSE21918 Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: sequencing data
Relations
SRA SRX020786
BioSample SAMN00013696

Supplementary file Size Download File type/resource
GSM545215_chr1.hits.txt.gz 9.3 Mb (ftp)(http) TXT
GSM545215_chr10.hits.txt.gz 4.3 Mb (ftp)(http) TXT
GSM545215_chr10_random.hits.txt.gz 3.5 Kb (ftp)(http) TXT
GSM545215_chr11.hits.txt.gz 4.0 Mb (ftp)(http) TXT
GSM545215_chr11_random.hits.txt.gz 5.5 Kb (ftp)(http) TXT
GSM545215_chr12.hits.txt.gz 3.4 Mb (ftp)(http) TXT
GSM545215_chr13.hits.txt.gz 2.1 Mb (ftp)(http) TXT
GSM545215_chr13_random.hits.txt.gz 5.6 Kb (ftp)(http) TXT
GSM545215_chr14.hits.txt.gz 2.1 Mb (ftp)(http) TXT
GSM545215_chr15.hits.txt.gz 1.9 Mb (ftp)(http) TXT
GSM545215_chr15_random.hits.txt.gz 15.7 Kb (ftp)(http) TXT
GSM545215_chr16.hits.txt.gz 2.1 Mb (ftp)(http) TXT
GSM545215_chr16_random.hits.txt.gz 3.2 Kb (ftp)(http) TXT
GSM545215_chr17.hits.txt.gz 2.0 Mb (ftp)(http) TXT
GSM545215_chr17_random.hits.txt.gz 89.0 Kb (ftp)(http) TXT
GSM545215_chr18.hits.txt.gz 1.3 Mb (ftp)(http) TXT
GSM545215_chr18_random.hits.txt.gz 196 b (ftp)(http) TXT
GSM545215_chr19.hits.txt.gz 1.6 Mb (ftp)(http) TXT
GSM545215_chr19_random.hits.txt.gz 2.9 Kb (ftp)(http) TXT
GSM545215_chr1_random.hits.txt.gz 27.0 Kb (ftp)(http) TXT
GSM545215_chr2.hits.txt.gz 3.9 Mb (ftp)(http) TXT
GSM545215_chr20.hits.txt.gz 1.1 Mb (ftp)(http) TXT
GSM545215_chr21.hits.txt.gz 577.9 Kb (ftp)(http) TXT
GSM545215_chr21_random.hits.txt.gz 78.0 Kb (ftp)(http) TXT
GSM545215_chr22.hits.txt.gz 772.8 Kb (ftp)(http) TXT
GSM545215_chr22_h2_hap1.hits.txt.gz 10 b (ftp)(http) TXT
GSM545215_chr22_random.hits.txt.gz 3.9 Kb (ftp)(http) TXT
GSM545215_chr2_random.hits.txt.gz 5.5 Kb (ftp)(http) TXT
GSM545215_chr3.hits.txt.gz 2.6 Mb (ftp)(http) TXT
GSM545215_chr3_random.hits.txt.gz 6.8 Kb (ftp)(http) TXT
GSM545215_chr4.hits.txt.gz 2.1 Mb (ftp)(http) TXT
GSM545215_chr4_random.hits.txt.gz 11.6 Kb (ftp)(http) TXT
GSM545215_chr5.hits.txt.gz 2.1 Mb (ftp)(http) TXT
GSM545215_chr5_h2_hap1.hits.txt.gz 10 b (ftp)(http) TXT
GSM545215_chr5_random.hits.txt.gz 1.8 Kb (ftp)(http) TXT
GSM545215_chr6.hits.txt.gz 2.1 Mb (ftp)(http) TXT
GSM545215_chr6_cox_hap1.hits.txt.gz 10 b (ftp)(http) TXT
GSM545215_chr6_qbl_hap2.hits.txt.gz 10 b (ftp)(http) TXT
GSM545215_chr6_random.hits.txt.gz 25.4 Kb (ftp)(http) TXT
GSM545215_chr7.hits.txt.gz 1.8 Mb (ftp)(http) TXT
GSM545215_chr7_random.hits.txt.gz 8.1 Kb (ftp)(http) TXT
GSM545215_chr8.hits.txt.gz 1.6 Mb (ftp)(http) TXT
GSM545215_chr8_random.hits.txt.gz 6.1 Kb (ftp)(http) TXT
GSM545215_chr9.hits.txt.gz 1.3 Mb (ftp)(http) TXT
GSM545215_chr9_random.hits.txt.gz 6.6 Kb (ftp)(http) TXT
GSM545215_chrM.hits.txt.gz 37.8 Kb (ftp)(http) TXT
GSM545215_chrX.hits.txt.gz 1.4 Mb (ftp)(http) TXT
GSM545215_chrX_random.hits.txt.gz 15.6 Kb (ftp)(http) TXT
GSM545215_chrY.hits.txt.gz 257.8 Kb (ftp)(http) TXT
GSM545215_master_table_01.out.txt.gz 7.4 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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