Illumina HiSeq 2000 sequencing; GSM1482491: Df(1)ED6957_F_Df(1)/w1118_rep_1 [Sample_100101]; Drosophila melanogaster; RNA-Seq

Df(1)ED6957_F_Df(1)/w1118_rep_1 [Sample_100101]

To measure the response to gene dose, we performed mRNA-Seq of fly heads with molecularly defined deletions constructed from DrosDel deficiency lines (Ryder et al. Genetics 2007, 177(1):615-29) on the Illumina HiSeq 2000 platform. Overall design: We performed single-end next-generation sequencing (RNA-Seq) on poly-A+ RNA extracted from adult female and male heads in biological triplicate. Besides wildtype females (XX) and males (XY) that were heterozygous for deletions, we also sequenced females that were transformed into males (XX males) by using mutations in the sex determination gene transformer-2 (tra2). The original lines with deletions, including 22 deletions on the chromosome X and 12 deletions on the chromosome 3L, were from the DrosDel project. The diploid controls without DrosDel deletions were derived from w1118 (the parental line of DrosDel stocks) or Oregon-R Strain. We sequenced a total of 249 samples.

RNA-Seq of female, male, and sex-transformed Drosophila melanogaster heads from flies heterozygous for deletions on chromosome X and 3L

Submitted by Gene Expression Omnibus on 27-MAR-2015

We used RNeasy 96 Kit (Qiagen, Valencia, CA) to extract total RNA from a pool of 10 dissected heads for each sample. Frozen heads were added to 2ml Axygen 96-well plates (Corning, Life Sciences, Union City, CA) pre-loaded with 200 µL 1mm glass beads (Biospec Products, Inc., Bartlesville, OK) and 200 µL RLT Buffer (Qiagen, Valencia, CA). After covering the plates with Axygen sealing mats, we homogenized the samples with Mini-BeadBeater-96 (Biospec Products Inc., Bartlesville, OK). Manual purification was carried out using QIAvac 96 vacuum manifold (Qiagen, Valencia, CA). We followed TruSeq RNA Sample Preparation V2 high-throughput protocol (Illumina Inc., San Diego, CA) to construct RNA-seq libraries from polyA+ RNA. For each sample, 100ng of total RNA was used as input in the initial step and 10pg ERCC spike-in RNA (Zook et al. Plos ONE 2012, 7(7): e41356., pool 78A) was added to the Elute, Prime, Fragment Mix in the "Purify and fragment mRNA” step.

RNA-Seq of female, male, and sex-transformed Drosophila melanogaster heads from flies heterozygous for deletions on chromosome X and 3L

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