|
| Status |
Public on May 21, 2010 |
| Title |
AGO1 |
| Sample type |
SRA |
| |
|
| Source name |
cultured cells, AGO1
|
| 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: AGO1
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 AGO1. Protein was immunoprecipitated by anti-FLAG antibody, Sigma F1854.
Sample: Length of RNA-segment (nt) used for cDNA generation
Ago1: 19-24
Processed data from AGO1 library.
|
| 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 |
SRX020783 |
| BioSample |
SAMN00013693 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM545212_chr1.hits.txt.gz |
5.9 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr10.hits.txt.gz |
2.7 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr10_random.hits.txt.gz |
2.3 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr11.hits.txt.gz |
2.5 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr11_random.hits.txt.gz |
2.9 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr12.hits.txt.gz |
2.3 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr13.hits.txt.gz |
1.5 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr13_random.hits.txt.gz |
4.0 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr14.hits.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr15.hits.txt.gz |
1.3 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr15_random.hits.txt.gz |
10.2 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr16.hits.txt.gz |
1.3 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr16_random.hits.txt.gz |
2.3 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr17.hits.txt.gz |
1.3 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr17_random.hits.txt.gz |
66.9 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr18.hits.txt.gz |
942.9 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr18_random.hits.txt.gz |
141 b |
(ftp)(http) |
TXT |
| GSM545212_chr19.hits.txt.gz |
1.1 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr19_random.hits.txt.gz |
2.0 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr1_random.hits.txt.gz |
21.4 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr2.hits.txt.gz |
2.7 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr20.hits.txt.gz |
748.0 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr21.hits.txt.gz |
400.5 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr21_random.hits.txt.gz |
41.7 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr22.hits.txt.gz |
474.0 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr22_h2_hap1.hits.txt.gz |
10 b |
(ftp)(http) |
TXT |
| GSM545212_chr22_random.hits.txt.gz |
2.5 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr2_random.hits.txt.gz |
2.8 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr3.hits.txt.gz |
1.9 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr3_random.hits.txt.gz |
4.9 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr4.hits.txt.gz |
1.6 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr4_random.hits.txt.gz |
7.2 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr5.hits.txt.gz |
1.6 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr5_h2_hap1.hits.txt.gz |
10 b |
(ftp)(http) |
TXT |
| GSM545212_chr5_random.hits.txt.gz |
1.5 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr6.hits.txt.gz |
1.6 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr6_cox_hap1.hits.txt.gz |
10 b |
(ftp)(http) |
TXT |
| GSM545212_chr6_qbl_hap2.hits.txt.gz |
10 b |
(ftp)(http) |
TXT |
| GSM545212_chr6_random.hits.txt.gz |
16.3 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr7.hits.txt.gz |
1.3 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr7_random.hits.txt.gz |
5.3 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr8.hits.txt.gz |
1.1 Mb |
(ftp)(http) |
TXT |
| GSM545212_chr8_random.hits.txt.gz |
3.9 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr9.hits.txt.gz |
972.1 Kb |
(ftp)(http) |
TXT |
| GSM545212_chr9_random.hits.txt.gz |
4.9 Kb |
(ftp)(http) |
TXT |
| GSM545212_chrM.hits.txt.gz |
24.2 Kb |
(ftp)(http) |
TXT |
| GSM545212_chrX.hits.txt.gz |
1.1 Mb |
(ftp)(http) |
TXT |
| GSM545212_chrX_random.hits.txt.gz |
13.5 Kb |
(ftp)(http) |
TXT |
| GSM545212_chrY.hits.txt.gz |
170.6 Kb |
(ftp)(http) |
TXT |
| GSM545212_master_table_01.out.txt.gz |
7.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
