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Sample GSM1180174 Query DataSets for GSM1180174
Status Public on Dec 25, 2013
Title E14.JARID2.NT.115k.b1
Sample type SRA
 
Source name E14 embyonic stem cells
Organism Mus musculus
Characteristics cell type: E14 embyonic stem cells
ip antibody: JARID2
antibody source: in-house
antibody lot #: PC-11
excised band size: 110–140 kDa
limited rnase treatment: no
Treatment protocol cells were pulsed with either 100 µM 4-SU for 16h (JARID2 PAR-CLIP) or 500 µM 4-SU for 2h (EZH2 PAR-CLIP)
Growth protocol E14 cells were grown in standard ESC conditions including 15% ESC certified serum
Extracted molecule total RNA
Extraction protocol Whole cell extracts were obtained by incubating the cells for 10 min at 37C 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 in the same CLIP buffer for 1 hour at 4C, after which, when required, the extracts were treated with various concentration of RNAse A + T1 cocktail (Ambion) for 5’ at 37C. Immunocomplexes were recovered by adding protein G-coupled dynabeads (Life technologies) for 45 min at 4C. Contaminating DNA was removed by treating the beads with Turbo DNase (2U in 20 μl). Crosslinked RNA was labeled by successive incubation with 5U Antarctic phosphatase (New England Biolabs) and 5U T4 PNK (New England Biolabs) in presence of 10 μCi [γ-32P] ATP (PerkinElmer, MA). Labeled material was resolved on 8% bis-tris gels, transferred to nitrocellulose membranes and exposed to autoradiography films for 1–24 hours.
100 pmol of a 3’-blocked DNA adapter were ligated to the RNA after dephosphorylation and before 5’ labeling by incubating the beads with T4 RNA ligase 1 (New England Biolabs) for 1 hour at 25C. After autoradiography, bands of interest were excised and the RNA eluted from the membrane by treating with proteinase K for 30’ at 37C and then proteinase K in presence of 3.5M urea for 30’ at 55C. Custom designed 5’ RNA adapters were ligated, the products size-selected on polyacrylamide or agarose gels.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
 
Description crosslinked RNA
Data processing Library strategy: PAR-CLIP-Seq
3' adapter (TTGATATAAATAGTGCCCATGGATC) trimmed with fastx_clipper
mapped to the mm9 genome using BOWTIE allowing for up to two mismatches and removing optical/PCR duplicates
putative RNA-binding sites identified with PARalyzer software (Corcoran et al., 2011) requiring at least two T->C conversions per RCS
Genome_build: mm9
Supplementary_files_format_and_content: BED files containing all unfiltered RCSs
 
Submission date Jul 03, 2013
Last update date Feb 04, 2022
Contact name Roberto Bonasio
Organization name University of Pennsylvania
Department Cell and Developmental Biology
Lab Bonasio
Street address 3400 Civic Center Blvd - SCTR 9-111
City Philadelphia
State/province PA
ZIP/Postal code 19104
Country USA
 
Platform ID GPL13112
Series (2)
GSE48517 PAR-CLIP for JARID2 in mouse embryonic stem cells
GSE48518 Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin
Relations
BioSample SAMN02225362
SRA SRX317614

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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