GSM2460712: mRNA X^XYohio ovary Replica 2; Drosophila melanogaster; RNA-Seq
Total RNA was isolated with Trizol. In order to check the quality of the RNA extracts, samples were run in an 1% TAE-Agarose gel and integrity of the mRNA was estimated based on the sharpness of the ribosomal RNA bands. Samples that did not meet this quality control were re-extracted. Concentration of all samples was accessed with Qubit 3.0 Fluorometer (Thermo Fisher Scientific). For mRNA-Seq, total RNA was subjected to mRNA isolation with magnetic beads linked to poly-T tails (Dynabeads® mRNA DIRECT™ Purification Kit) for two cycles. The quality of mRNA preparations was accessed with Agilent 2100 Bioanalyzer system (Agilent Technologies). Samples with intact mRNA and less than 15% of rRNA contamination were subjected to library synthesis using the automated Apollo 324™ NGS Library Prep system with PrepX RNA-Seq kit (Wafergen). Final product of each library was subjected to 15 cycles of PCR enrichment in reactions containing one barcode for each sample. Final libraries were purified with magnetic beads to an average size of 350 bp and checked with Agilent 2200 TapeStation (Agilent Technologies). Samples were normalized with Qubit 3.0 Fluorometer, and twelve libraries were combined in a single mix. Multiplexed mix was submitted to 150 cycles of paired-end sequencing (2x150bp) using HiSeq 2500 (Illumina). Two biological replicates were sequenced for each sample.
GEO Accession
GSM2460712