GSM6125436: S1_G2_M_R2; Drosophila melanogaster; RNA-Seq
Total RNA was extracted using Qiazol (Qiagen, Hilden, Germany), followed with phenol-chloroform extraction, isopropanol precipitation, and DNase digestion (Qiagen, Hilden, Germany).  Qiagen RNeasy MinElute Cleanup kits (Qiagen, Hilden, Germany) were used to purify RNA according to the manufacturer's instructions.  Total RNA yield was measured using a Nanodrop 8000 (Thermo Scientific, Waltham, MA).  We used Agilent Bioanalyzer RNA chips on the 2100 Bioanalyzer system (Agilent, Santa Clara, CA) according to the manufacturer instructions to visually inspect electrophoregrams for RNA degradation. PolyA-selected stranded mRNA libraries were constructed from 1 µg total RNA using Illumina TruSeq Stranded mRNA Sample Prep Kits (Illumina, San Diego, CA).  We followed the manufacturer's protocol with the exception of the PCR amplification. The PCR amplification was performed for 10 cycles rather than 15 in order to reduce potential over-amplification.  We applied unique barcode adapters to each library. Libraries were pooled for sequencing.  To achieve a minimum of 38 million 126-base read pairs, the pooled libraries were sequenced on multiple lanes of an Illumina HiSeq2500 (Illumina, San Diego, CA) using version 4 chemistry.