Illumina HiSeq 2000 sequencing; GSM1506809: Df(2L)ED3/+_female_rep1; Drosophila melanogaster; RNA-Seq

Df(2L)ED3/+_female_rep1

We performed mRNA transcriptional profiling on 99 hemizygotic lines (Df/+) from the DrosDel project covering 68% of chromosome 2L, in order to understand how changes in gene copy number affect overall transcriptome. Overall design: We performed RNA-Seq analysis on 396 pools of 15-25 adult flies each. Samples include males or females from 99 different genotypes in duplicate. Differential gene expression was analyzed separately for each sex, by comparing each genotype with the remaining 98.

Expression profiling pooled Drosophila melanogaster heterozygous for deletions on Chromosome 2L

Submitted by Gene Expression Omnibus on 01-FEB-2016

Flies were homogenized in 500 ul of RNAlater solution (Life Technologies, Grand Island, NY, USA) for 90 seconds in 96 deep well plates with 3mm tungsten carbide beads (Qiagen, West Sussex, UK) using a Qiagen Tissue Lyser II (Qiagen, West Sussex, UK). Extraction of total RNA was pefromed using RNeasy 96 kit (Qiagen, Valencia, CA, USA) based on the manufacturer's guide (Protocol for Isolation of Total RNA from animal cells using QIAvac96 vacuum manifold, Cat#19504). A minor modification is made in order to dilute RNAlater solution, so instead of mixing 150 ul of RLT buffer (Qiagen, Cat#79216) and 150 ul of 70% ethanol, we mixed 150 ul of RLT buffer, 120 ul of 100% ethanol, and 30 ul of lysate in RNAlater and applied to the extraction columns. Libraries were prepared using Illumina's (San Diego, CA, USA) TruSeq RNA sample preparation kit v2 - set A and B (Cat # RS-122-200). RNA amount was quantified using RiboGreen kit (Life Technology, Grand Island, NY, USA). 100 ng of RNA was used as input to follow the 1/2 scaled protocol from the original manufacturer's High Sample Protocol (Illumina, Part# 15026495 Rev. C). We added 50 pg of external RNAs as spike-in (Zook et al, 2012 PLoS One) during the 8 minute of RNA fragmentation step (Note. For samples with IDs of #1 to # 24, 1ng of spike in was added). The constructed libraries were sequenced on the HiSeq 2000 (Illumina, San Diego, CA, USA).

Expression profiling pooled Drosophila melanogaster heterozygous for deletions on Chromosome 2L

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