title: GSM1650072: Dsec female TW rep2; Drosophila sechellia; RNA-Seq
library_construction_protocol: About 300-700 pairs of fly antennae of each sex from each species were collected for total RNA isolation.  The antennae resected each day were preserved in ~ 50-100 ul Trizol (Life Technology) and stored at -80 °C before further processing.  Right before the RNA isolation, we spun down antennae preserved in Trizol at the max speed for 3 min at 4 °C and put each sample into one MagNA Lyser Green Beads tube (Roche). The RNA isolation protocol we developed combined steps from conventional Trizol extraction and RNeasy Micro Elute Kit (QIAGEN, Inc.) with some modifications to obtain high yield and quality of total RNA.  Fly antennae were disrupted with MagNA Lyser (Roche) at 7000 rpm for 15 seconds each time and repeated for 4-5 times until the tissue was almost invisible.  After 15 sec, we cooled down the lysate on ice for 10 sec to prevent RNA degradation.  Tissue lysate was transferred to a new RNase free Eppendorf tube.  We used about 400 ul Trizol to rinse the beads of each sample and combine this 400 ul Trizol with the tissue lysate for the RNA isolation.  Based on the suggestion of the Trizol standard protocol, 200 microliters of chloroform per microliter of tissue lysate were added to the lysate and mixed well by shaking vigorously for 15 sec and set 2-3 min at room temperature.  The aqueous phase was separated by centrifuging the lysate at the maximum speed at 4 °C for 15 min and carefully transferred to a new Eppendorf tube after centrifuging. We added 1 volume of 5 µl Carrier RNA (QIAGEN, Inc.) and one microliter of 70% EtOH to the aqueous phase and mixed well by inverting the tubes carefully.  To obtain greater amount of RNA, we kept the samples at -80 °C for overnight to aid in RNA precipitation.  RNA isolation followed manufacturer’s protocol of the RNeasy Micro Elute Kit with increased volumes (700 ul) of RPE buffer and 80% ethanol to wash RNA.  Genomic DNA was removed by applying on-column DNase I treatment at room temperature for 15 min. For paired-end mRNA-seq library preparation, we used Illumina TrueSeq kits.  A total of 10 µg total RNA was used for mRNA enrichment by oligo-dT beads followed by cation-catalyzed fragmentation for 4 minutes at 94˚C.  The mRNA fragments were then converted into double stranded cDNA by random priming followed by end repair.  The fragmented cDNAs from each sex of each species were then ligated to the barcoded paired-end adaptors and subjected to 15 cycles of PCR amplification and purified by Ampure beads (Beckman Agencourt). The absolute concentrations of the libraries were determined by Qubit fluorometry (Invitrogen) and BioAnalyzer High Sensitivity DNA Kit (Agilent).  We combined barcoded cDNA from 6 samples and loaded them in 3 sequencing lanes of flow cell, and paired-end 2*100nt sequencing was conducted on Illumina HiSeq2000.