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Status |
Public on Mar 29, 2013 |
Title |
short_capRNA__1_tech |
Sample type |
SRA |
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Source name |
mixed embryos
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Organism |
Caenorhabditis elegans |
Characteristics |
developmental stage: mixed embryos strain: N2 (wild-type)
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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).
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
single-end
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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
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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
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Organization name |
University of Cambridge
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Department |
WT/CRUK Gurdon Institute
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Street address |
Tennis Court Road
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City |
Cambridge |
ZIP/Postal code |
CB2 1QN |
Country |
United Kingdom |
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Platform ID |
GPL13657 |
Series (1) |
GSE42819 |
The landscape of RNA polymerase II transcription initiation in C. elegans reveals a novel regulatory architecture |
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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 Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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