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Sample GSM1050559 Query DataSets for GSM1050559
Status Public on Mar 29, 2013
Title short_capRNA__1_tech
Sample type SRA
 
Source name mixed embryos
Organism Caenorhabditis elegans
Characteristics developmental stage: mixed embryos
strain: N2 (wild-type)
Growth protocol Worms were grown in liquid culture and two million adults bleached to obtain mixed staged embryos as previously described (Stiernagle, 2006). Nuclei were isolated from embryos using the method of Ooi et al (Ooi et al., 2010). Nuclei were further purified by centrifugation through a 1.8M sucrose cushion in 10 mM Tris pH 7.5, 10 mM MgCl2. RNA was extracted from the purified nuclei using Tripure (Roche).
Extracted molecule nuclear RNA
Extraction protocol To prepare short capRNA-seq libraries, we followed the published method by Nechaev et al (Nechaev et al., 2010) with minor modification. In brief, total nuclear RNA (20 to 25 ug) was run on a polyacrylamide gel and a size range of 20 to 100 nt extracted. To enrich for capped RNA, the purified short nuclear RNA was incubated with 5’ to 3’ RNA nuclease, Terminator (Epicentre), to deplete non-capped RNA. The enriched short capped RNA was cloned and PCRed using Illumina short RNA-seq adapters according to the manual instruction. To construct long capRNA-seq libraries, total nuclear RNA was incubated with DNAse I (Ambion) to remove genomic DNA contamination followed by Qiagen Mini-Elute columns (cut-off size >200 nt RNAs). These cleaned-up “long” nuclear RNAs were treated with Terminator nuclease to enrich for capped RNA. Strand specific long capRNA libraries were made using the dUTP replacement method (Levin et al., 2010, Sultan et al., 2012) and Illumina paired end adapters.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Description single-end
Data processing Reads were aligned to the reference sequence using bwa-0.5.9 then sorted and indexed using samtools
Short-cap reads with quality scores >=10 were extracted, split by strand, and the numbe of tag 5' ends aligning at a given site were counted
Pairs of long-cap reads with quality scores >= 10 were extracted.
Genome_build: WS220/ce10
Supplementary_files_format_and_content: bigWig files of of cap start positions for short-cap libraries bigBed files of read-pair mappings for long-cap libraries
 
Submission date Dec 10, 2012
Last update date May 15, 2019
Contact name Thomas A Down
E-mail(s) thomas.down@gurdon.cam.ac.uk
Organization name University of Cambridge
Department WT/CRUK Gurdon Institute
Street address Tennis Court Road
City Cambridge
ZIP/Postal code CB2 1QN
Country United Kingdom
 
Platform ID GPL13657
Series (1)
GSE42819 The landscape of RNA polymerase II transcription initiation in C. elegans reveals a novel regulatory architecture
Relations
SRA SRX209304
BioSample SAMN01828200

Supplementary file Size Download File type/resource
GSM1050559_rep1t-minus.bw 1.7 Mb (ftp)(http) BW
GSM1050559_rep1t-plus.bw 1.7 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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