Cylas puncticollis RNA-seq
Libraries were constructed at the North Carolina State University Genomic Sciences Laboratory following the Illumina TruSeq v2 mRNA sample preparation protocol. Starting RNA is first checked on the Agilent 2100 Bioanalyzer using a RNA Pico Chip for quality and concentration. The Illumina mRNA library protocol uses a starting amount of 1ug of total RNA. Total RNA was purified using poly-T oligo-attached magnetic beads to isolate poly-A containing mRNA.  The mRNA was chemically fragmented using divalent cations and fragments were reverse transcribed into first strand cDNA using random primers. Second strand synthesis was carried out using DNA Polymerase I and RNaseH.   Ends were repaired on the double-stranded cDNA to convert overhangs into blunt ends, 3’ ends were adenylated, and adapters with indexes for multiplexing were ligated.  The library was amplified with a PCR reaction and then AmpureXP beads were used to remove small fragments and for sizing.  The final library was quantified using Agilent’s Bioanalyzer High Sensitivity DNA Chip prior to clustering on the Illumina cBot. Sequencing was performed on a HiSeq2000 and each sample was sequenced in one lane of a 100bp single-end run.