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Sample GSM914114 Query DataSets for GSM914114
Status Public on May 31, 2012
Title Nascentseq_Fly_yw_R1
Sample type SRA
 
Source name head, yw, nascent RNA
Organism Drosophila melanogaster
Characteristics strain: yellow white (yw)
tissue: head
age: 3-4 days
gender: male
Treatment protocol Drosophila flies were entrained to 3-4 days of 12hr light : 12hr dark cycles.
Extracted molecule total RNA
Extraction protocol Fly heads were isolated with brass sieves. Fly head nuclei were purified and Nascent RNA was extracted as described in Wuarin and Schibler, 1994 (PMID 7523861). Nascent RNA was extracted from the NUN pellet with Invitrogen TRIzol Reagent using the manufacturer’s protocol. Nascent RNA replicate set 1 was ribosomal RNA depleted as described in Khodor and Rosbash, 2011. All Nascent RNA samples were depleted of mRNA by two rounds of pA depletion using Invitrogen Dynabeads Oligo dT using the manufacturer’s protocol. Sequencing library construction was performed by using the standard Illumina protocol for RNA starting with 100 nanograms of Nascent RNA.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Description Nascent RNA.
Small-scale prep from 40-45 heads.
Data processing Small-scale prep base calls performed using CASAVA v1.8.
Nascent-seq reads were aligned to the dm3 genome assembly using tophat with the following parameters: "-m 1 -F 0 --microexon-search --no-closure-search -G exon20110421.gtf --solexa1.3-quals -I 50000". yw, FM7a, ADAR0 male sequences were aligned with the following parameters: "-m 1 -F 0 --microexon-search --no-closure-search -G exon20110421.gtf --solexa-quals -I 50000". The gtf file corresponds to UCSC genome table gene annotation from the RefSeq table.
Base frequencies were calculated within exons and introns of UCSC annotation. Genes with multiple isoforms were flattened, where overlapping exons generate one exon. Base positions with one or more Gs in the nascent datasets and zero Gs in the sequenced genomic DNA were identified. We required that editing sites occur in at least 5 of 6 samples within each set of replicate time points, for a total of 10 of 12 independent occurrences for each site. To avoid potential mismapping of reads at splice junctions by Tophat, we required that edited sites occur in at least one of the two middle quadrants of at least one read. Intronic sites that occurred within 10 bases of an annotated splice site were also discarded. We applied a similar approach to the yw pA-seq data, with the exception that we required that the editing site occur in both samples (2 of 2). Editing levels were then calculated for the sites found in the nascent analysis. For reproducibility of the nascent level, we pooled the editing counts for each replicate set of 6 time points together and calculated the percent editing level. The final percent editing level was determined by pooling the editing counts for all 12 samples and dividing by the total pooled counts of the 12 samples. Editing was similarly calculated for the yw pA-seq data and the small-scale Nascent-seq data by pooling both replicate samples. The editing level for the Cs pA-seq data was calculated by pooling all 12 samples. The exon and intron editing sites identified in the 12 Nascent-seq replicates were scanned in the small-scale ADAR0, FM7a, yw Nascent-seq data. We required editing to occur in both replicates of the FM7a and yw data, and sites with zero sequence coverage in any sample were not considered.
Processed data (available as a supplementary file on the Series record): Editing frequencies.
Genome_build: dm3
 
Submission date Apr 12, 2012
Last update date May 15, 2019
Contact name Joseph Rodriguez
Organization name Brandeis University
Department Biology
Lab Rosbash
Street address 415 South Street
City Waltham
ZIP/Postal code 02453
Country USA
 
Platform ID GPL13304
Series (1)
GSE37232 Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila
Relations
Reanalyzed by GSM3277400
SRA SRX141945
BioSample SAMN00854976

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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