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Sample GSM1490023

Query DataSets for GSM1490023

Status

Public on Apr 23, 2017

Title

CY21B3_RNA

Sample type

SRA

Source name

head

Organism

Drosophila yakuba

Characteristics

strain: CY21B3
tissue: head
developmental stage: adult

Extracted molecule

total RNA

Extraction protocol

Total RNA was obtained from each Drosophila yakuba strain. Approximately 50 fly heads were used to obtain total RNA (5 µg) using TRIzol reagent (Life) as per manufacturer’s instruction. RNA integrity was evaluated on the BioAnalyzer (Aligent). Oligo(dT) selection was performed by using Dynal magnetic beads (Invitrogen) according to the manufacturer's protocol. The mRNA was fragmented by addition of 5x fragmentation buffer (200 mM Tris acetate, pH 8.2, 500 mM potassium acetate and 150 mM magnesium acetate), heated at 94 °C for 90 seconds, and then transferred to ice and run over a Sephadex-G50 column (USA Scientific) to remove the fragmentation ions.The fragmented mRNA was incubated in 38 µl CE solution (50% (v/v) ethanol, 1.1 M triethylammoniumacetate (pH 8.6)) with 1.6 M acrylonitrile at 70 °C for 1 hr (ICE treatment). A parallel mock treatment was preformed without 1.6 M acrylonitrile. The RNA was purified with ethanol precipitation and reverse transcribed with random hexamers, added to prime first-strand reverse transcription according to the manufacturer's protocol (Invitrogen cDNA synthesis kit). After the first strand was synthesized, a custom second-strand synthesis buffer (Illumina) was added, and dNTPs, RNase H and Escherichia coli polymerase I were added to nick translate the second-strand synthesis for 2.5 h at 16 °C. The reaction was then cleaned up on a QiaQuick PCR column (Qiagen) and eluted in 30 µl EB buffer (Qiagen).HiSeq reads were sequenced as single-ended 50-mers and NextSeq reads were sequenced as 86-mers. The reads were mapped onto the Drosophila melanogaster genome (BDGP R5/dm3) or Drosophila yakuba genome (WUGSC 7.1/droYak2) using TopHat version 2.0.6 (Kim 2013) / Bowtie version 2.0.2 (Langmead 2012) using the following options: --read-realign-edit-dist 0 --read-edit-dist 4 --read-mismatches 4 -G <gene annotation file> -x 1 -g 1. Unmapped reads were allowed to remap using bsmap version 2.74 (Xi 2009) using the following options: -v 3 -M GA.
The RNA was purified with ethanol precipitation and reverse transcribed with random hexamers, added to prime first-strand reverse transcription according to the manufacturer's protocol (Invitrogen cDNA synthesis kit). After the first strand was synthesized, a custom second-strand synthesis buffer (Illumina) was added, and dNTPs, RNase H and Escherichia coli polymerase I were added to nick translate the second-strand synthesis for 2.5 h at 16 °C. The reaction was then cleaned up on a QiaQuick PCR column (Qiagen) and eluted in 30 µl EB buffer (Qiagen)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

mock treatment

Data processing

An initial set of sites were obtained using samtools (Li 2009) version 0.1.19 requiring variants to be seen in at least two RNA-seq replicates and a minimum editing rate of 5%. We also required the following for at least one replicate: a p-value less than 0.01 for the variant frequency between the ICE and RNA samples, less coverage in the ICE-seq than the RNA-seq, and a lower variant frequency in the ICE-seq than the RNA-seq. The Storer-Kim method was used to determine the p-value because it is considered one of the better statistical tests, in terms of power and Type I error, particularly for small and/or unequal sample sizes. We limited ourselves to A-to-G, T-to-C, G-to-A, and C-to-T variants because of the error bias in the NextSeq. The A-to-G and T-to-C variants represented our potential set of RNA editing sites (T-to-C variants were included because we used an unstranded library protocol). The G-to-A and C-to-T variants were used as a negative control because these types of variants had a comparable error profile and drosophila do not have C-to-U RNA editing. We computed the IDR for each site using the change in the number of G’s from RNA-seq to ICE-seq as the signal value. The sites were sorted by IDR and a 0.85 fraction of A-to-G/T-to-C sites were used to determine a cutoff threshold. Of the remaining sites, we removed sites outside gene boundaries, sites that were not A-to-G in the direction of transcription, and sites in repetitive regions. We sequenced two biological replicates of RNA-seq libraries derived from Drosophila melanogaster heads. The libraries were sequenced on a HiSeq 2500 and NextSeq 500. The resulting reads were normalized for read number and read length and mapped onto the dm3 assembly using tophat2 (Kim 2013) version 2.0.6. Variants were called using samtools mpileup with any at least 1 read supporting a variant. We reasoned that true biological variants and mapping errors were shared between the two sequencers while sequencing errors should be in the outersects. Variants that were sequencer specific were determined to be sequencing errors. Gene ontology analysis was done using WebGestalt (Wang 2013). A significance level of 0.01 adjusted with the Benjamini-Hochberg procedure was used. A minimum of 5 genes per category was required.
Genome_build: dm3/droYak2
Supplementary_files_format_and_content: bed files containing RNA editing sites or other variants

Submission date

Aug 27, 2014

Last update date

May 15, 2019

Contact name

Eddie Park

E-mail(s)

eddiep@uci.edu

Phone

9498248393

Organization name

UCI