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| Status |
Public on Jan 20, 2014 |
| Title |
adr2- knockout worms |
| Sample type |
SRA |
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| Source name |
BB20 adr-2(ok735)
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| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: whole worms
genotype: BB20 adr-2(ok735)
genetic background: N2
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| Treatment protocol |
The injection mix used for generating transgenics contained the following: 1ng/μl of the transgene of interest, 20ng/μl of the dominant marker, and 79ng/μl of 1kb DNA ladder (NEB)
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| Growth protocol |
Transgenic worm lines were generated by microinjection into the gonads of young adult worms of the appropriate genetic background
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent.
RNA libraries were prepared for strand specific RNA sequencing using the published protocol (Parkhomchuk et al. Nucleic Acid Research. 2009 37(18):e123)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
RNA extracted by Trizol reagent and isolated with cloroform.
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| Data processing |
ssRNAseq: The adr-1(-);adr-2(-) sample was sequenced on one lane of Illumina's HiSeq 2000 yielding 216 million single-end 76nt reads. Each other sample was sequenced on a lane of Illumina GAII yielding between 37 and 42 million reads of the same type. Illumina Casava1.8.2 software used for basecalling.
Mapping: Sequenced reads were mapped to the C. elegans reference genome (ce10) using TopHat aligner (version 2.0.6) allowing only uniquely-mapped reads with up to two mismatches each with command line options -Mx 1 and -N 2
Variant calling: sites with RNA-DNA differences were identified by SAMtools mpileup (version 0.1.18) tallying up to 1000 alignments per site. Additional command line options used were -D -I and -g.
Site filters: Annotated SNPs were obtained from Illumina's iGenomes collection for C. elegans (ce10) and unannotated variants were extracted from the adr-1(-);adr-2(-) RNA-Seq dataset. These genomic variants were filtered from the putative sites in all other strains reducing the number of false-positive predictions.
Read filters: Each read aligned to one the remaining putative sites was filtered out if: a) it was a suspected PCR duplicate, according to SAMtools rmdup (version 0.1.18); b) it had a junction overhang < 10nt according to its SAMtools CIGAR string; c) it had > 1 non-A2G or non-C2T mismatch or any short indel, per its MD tag; d) it had a mismatch less than 25nt away from either end of the read
Identify sites: Putative RNA editing sites were identified from A2G variants on the sense strand and T2C variants on the antisense strand that were covered by more than 5 reads which passed the filters in step 5, including the stringent 25nt threshold for filter 5d).
Quantify sites: The extent of editing at each site and our confidence in that prediction were quantified by a novel extension of the classical Bayesian model used for genomic variants, which is described in more detail in the next section.
To increase the accuracy and confidence of our predictions, we used additional reads from the relaxed version of filter 5d) that overlap the sites identified in step 6. Moreover, we dropped sites that exhibited editing in 100% of the reads (suggesting a genomic variant not filtered out by step 4) and those with very low editing (less than 10%), which would have been hard to distinguish from sequencing errors.
The predicted RNA editing sites from each strain were characterized according to their position in annotated genic regions (introns, exons, 3'/5' UTRs, etc.) and according to their overlap with other strains.
Supplementary_files_format_and_content: tab-delimited text file containing #READS and %EDITING values for each editing site
Genome_build: ce10
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| Submission date |
Oct 22, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Boyko Kakaradov |
| Organization name |
UCSD
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| Department |
Bioinformatics
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| Lab |
Yeo Lab, SCRM 3115
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| Street address |
2880 Torrey Pines Scenic Dr.
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL9269 |
| Series (1) |
| GSE51556 |
The dsRBP and inactive editor, ADR-1, utilizes dsRNA binding to regulate A-to-I RNA editing across the C. elegans transcriptome |
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| Relations |
| BioSample |
SAMN02380470 |
| SRA |
SRX366863 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1248099_Sample_ADR12.conf0.995.txt.gz |
151 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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