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Sample GSM1296149 Query DataSets for GSM1296149
Status Public on Mar 18, 2014
Title U2OS.CTCF.m
Sample type SRA
Source name U2OS
Organism Homo sapiens
Characteristics cell line: osteosarcoma U2OS cell line
antibody: anti-CTCF (Abcam, cat# ab70303, lot#s GR98618-4 and GR27477-7)
Treatment protocol PAR-CLIP was performed as described with some modifications (Bonasio, et al 2013 sub.) Briefly, U2OS cells were grown under standard conditions and pulsed with 400 µM 4-SU (Sigma) for 16–24 h. After washing the plates with PBS, cells were crosslinked with 400 mJ/cm2 UVA (365 nm) using a Stratalinker UV crosslinker (Stratagene, CA).
Growth protocol U2OS cells were grown under standard conditions
Extracted molecule total RNA
Extraction protocol Whole nuclear lysates (WNL) were obtained by fractionating cytoplasm and nuclei by a standard method (Dunham, 1985), then nuclei were incubated for 10 min at 37ºC in an appropriate volume of CLIP buffer (20 mM HEPES pH 7.4, 5 mM EDTA, 150 mM NaCl, 2% lauryldimethylbetaine) supplemented with protease inhibitors, 20 U/ml Turbo DNase (Life technologies), and 200 U/ml murine RNase inhibitor (New England Biolabs). After clearing the lysate by centrifugation, immunoprecipitations were carried out using 1 mg of WNL, appropriate antibody and protein G-coupled dynabeads (Life technologies) in the same CLIP buffer, ON at 4ºC, after which, contaminating DNA was removed by treating the beads with Turbo DNase (2 U in 20 µl). Then the crosslinked RNA was labeled by successive incubation with 5 U Antarctic phosphatase (New England Biolabs), ligation of a 3′-blocked DNA adapter (100 pmol/μl) and incubation with 5U T4 PNK (New England Biolabs) in the presence of 10 µCi [γ-32P] ATP (PerkinElmer, MA). Labeled material was resolved on 8% bis-tris gels, transferred to nitrocellulose membranes and visualized by autoradiography.
Libraries were prepared according to Illumina's instructions. Briefly, The band of interest was excised and RNA eluted from the membrane by treatment with proteinase K (Roche, 4 mg/ml) for 30 min at 37 °C and then with proteinase K in the presence of 3.5 M urea for 30 min at 55 °C. After phenol/chloroform extraction, custom-designed 5′ RNA adapters were ligated, the products were size-selected on polyacrylamide gels, and libraries were amplified and sequenced on an Illumina HiSeq 2000 sequencing system.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Description Sample 3 (It is simply the merged file of the sequencing data from Samples 1 and 2).
library strategy: PAR-CLIP
Data processing Adapters were trimmed & sequences mapped to hg19 with BOWTIE -m40 -v2 --best
Optical/PCR duplicates were removed & reads mapping to repetitive regions (repeatMasker) were removed
bigBed files were generated
Replicate experiments were merged & intersectBed utility was used to map read counts to ensembl annotation
Annotation was filtered for genes that contain at least 5 mapped reads to generate .TXT file
Genome_build: hg19
Supplementary_files_format_and_content: bigBed files containing PAR-CLIP reads & TXT file containing # reads mapping to each gene
Submission date Dec 20, 2013
Last update date May 15, 2019
Contact name Danny Reinberg
Phone 212-263-9036
Organization name NYU School of Medicine / HHMI
Street address 522 First Avenue, 2nd floor Rm 211
City New York City
State/province New York
ZIP/Postal code 10016
Country USA
Platform ID GPL11154
Series (1)
GSE53554 PAR-CLIP-seq reveals RNAs directly interacting with CTCF in human transformed cell line U2OS
BioSample SAMN02484442
SRA SRX397060

Supplementary file Size Download File type/resource
GSM1296149_U2OS.CTCF.m.ensembl.txt.gz 287.2 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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