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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