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Sample GSM2407067

Query DataSets for GSM2407067

Status

Public on Nov 22, 2019

Title

mip130 RNA rep1

Sample type

SRA

Source name

S2R+

Organism

Drosophila melanogaster

Characteristics

agent: mip130

Treatment protocol

RNAi treatments were carried out over 4 days, with cell harvesting on the end of the 4th day. At the start of each day, the media was aspirated from the sample and 30µg/ml of the desired dsRNA in FBS-free Sheilds & Sang insect media. This was followed by incubation at 25oC for 30 minutes, after which S&S insect media with 10% heat-inactivated FBS was added to achieve a final dsRNA concentration of 10µg/ml.

Growth protocol

Approximately 5x106 S2R+ cells were plated on 25cm flasks in Shields and Sang insect media with 10% heat-inactivated fetal bovine serum (FBS). All samples were passaged an identical number of times before use, and all were incubated at 25oC throughout growth and treatments.

Extracted molecule

total RNA

Extraction protocol

Chromatin (DNA) preparation: Cells were washed twice with 1X PBS, then gently permeabilized using 950µl spheroplast digestion buffer containing NP-40 (SDBN, 1M Sorbitol, 50mM NaCl, 10 mM Tris-HCL [pH7.5], 5mM MgCl2, 1mM CaCl2, 0.5mM Spermidine, 1mM β-Mercaptoethanol, 0.075% NP-40). Micrococcal Nuclease was added to a final concentration of 600U/ml, and the plate was gently rocked for 3 minutes at room temperature. The reaction was stopped with 100µl STOP solution (5% SDS, 250mM EDTA). DNA was extracted using 2 phenol/chloroform purifications, with RNase treatment (5µl, 10mg/ml, 30 minutes, 37oC) after the first purification. 100% ethanol containing 0.3M NaOAc was used to precipitate DNA.
RNA: Cells were harvested from 1x4cm area of plate after PBS washes (see chromatin protocol) but before digestion. Total RNA was harvested using the RNAeasy mini kit (Qiagen) as per manufacturer's guidelines.
NKD DNA: The genomic DNA extracted from a single fly was incubated with 22u/ml Mnase for 20 seconds. The reaction was stopped with 100µl STOP solution (5% SDS, 250mM EDTA). RNase treatment (5µl, 10mg/ml, 30 minutes, 37oC) was followed with DNA extraction using a phenol/chloroform purification. 100% ethanol containing 0.3M NaOAc was used to precipitate DNA.
DNA: All sequencing performed by Exeter University Sequencing service. Standard Illumina protocols were performed for samples being sequenced on a Illumina HiSeq 2000 machine running in paired-end 100bp mode. During size selection, primer dimers were removed and all other DNAs were used for sequencing.
RNA: All sequencing performed by Exeter University Sequencing service. Standard Illumina protocols were performed for samples being sequenced on a Illumina HiSeq 2000 machine running in paired-end 100bp mode. RNA was run through an oligo dT column prior to cDNA generation to filter for mRNA transcripts only

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Base calling was performed using the Phred algorithm, limiting base calls to a Q-score of ≥30
DNA sequence alignment was performed against the dm5.53 genome release and annotation (dos Santos et al. 2015) using Bowtie v1.1.1 with parameters -v 3 --trim3 14 --maxins 5000 --fr -k 1 -p 15 --sam
The .sam file was separated into chromosome specific files using the bash script chrgrep.sh (see supplementary files for all scripts)
The chromosome specific .sam files were processed using SAMparser.plx to extract DNA fragments of a user defined insert length range (or ISIZE). The output .txt files for replicates were pooled using the UNIX "cat" function.
The .sgr files were processed with histogram.plx, which calculated dyad frequency for individual 10bp bins across each chromosome. A 3 bin moving average is applied by histogram.pl for light smoothing of the data, which was output in .sgr format.
Scaler.plx was used on the .sgr output for the X chromosome using a scale factor of 1.5 to account for the detected copy number variations found in the S2R+ samples.
Dros_cat.sh was used to pool all of the chromosome specific files into a single genomic .sgr
RNA sequence alignment was performed against the dm5.53 genome release and annotation (dos Santos et al. 2015) using Tophat v2.0.12 with parameters -i 40 -p 8 -o -G --library-type fr-secondstrand
Quantification of mapped reads was performed by Cufflinks with parameters -o –u –b –g –p 8 --min-intron-length 40
Assemblies were merged using Cuffmerege with parameters -o -g –p 8 -s
Normalization of read abundance values across samples was performed using Cuffnorm, with parameters -o -L -p 8
Genome_build: dm5.53 (dos Santos et al. 2015)
Supplementary_files_format_and_content: .bedgraph files: generated from the genomic .sgr containing number of fragments of a particular size range (+/- 0.2 for 150 and 225 fragments, +/- 10bp for 110bp fragments) with dyads mapping to the bin.
Supplementary_files_format_and_content: FPKM_expression_table.xlsx: Excel spreadsheet containing FPKM values for each transcript across each sample.

Submission date

Nov 28, 2016

Last update date

Nov 22, 2019

Contact name

Helen White-Cooper

E-mail(s)

White-CooperH@cardiff.ac.uk

Organization name

Cardiff University

Street address

Museum Avenue