title: RNAseq of D. montana examing the effects of light and diapause
design:  The libraries were constructed following  the SOLiD total RNA-seq Kit (Whole Transcriptome library) protocol  The stepwise work flow:    1. RNA isolation Total RNA was isolated. The quality of the RNA was checked with Agilent's 2100 Bioanalyzer and the concentrations with Nanodrop ND-2000 spectrophotometer.  2.  Fragmentation of RNA Poly(A) RNA was isolated and precipitated with MicroPoly(A)PuristTM Kit. Poly(A) RNA was fragmented using RNaseIII, to convert the whole transcriptome sample to RNA of a size appropriate for SOLiD™ System sequencing. After cleanup using the PurelinkTM RNA Micro kit, fragmented RNA samples with sufficient yield and an appropriate size distribution were ready for preparation of amplified cDNA libraries. Quality of the fragmentation was checked with Bioanalyzer.  3. Hybridization and ligation to Adaptor Mix The fragmented RNA sample was next hybridized and ligated with the Adaptor Mix. The Adaptor Mix contains oligonucleotides with a single-stranded degenerate sequence at one end and a defined sequence required for SOLiD System sequencing at the other end.   4. Reverse transcription and size selection RNA population with ligated adaptors was reverse transcribed to generate single-stranded cDNA copies of the fragmented RNA molecules. cDNA was purified and size selected with two rounds of Agencourt® AMPure® XP Reagent.         5. cDNA library amplification (single- or multiplex) and final cleanup The size-selected cDNA was next amplified using 15 cycles of PCR. This step appends required terminal sequences to each molecule and generates sufficient template for SOLiD sequencing. After the PCR, the amplified cDNA was cleaned up using Agencourt® AMPure® XP Reagent. • For multiplexing , the SOLiD Transcriptome Multiplexing Kit (barcodes 1-16) was used.  6. Library quantitation and quality control Libraries were quantitated with two different methods; Qubit fluorometer (Invitrogen) and quantitative PCR to warrant the accuracy.   -Quantitative PCR (qPCR) SOLiD Library TaqMan Quantitation Kit was used for determining the molar concentration of amplified template in a SOLiD library. The qPCR provides high level of specificity required and can accurately measure extremely low quantities of DNA. For optimal amplification during the emulsion PCR step, precise quantification of the input library is absolutely critical.  In qPCR, the standard and unknown library template are amplified using two sequence-specific primers with a TaqMan fluorogenic probe labeled with FAM™ dye and a dye quencher. A fluorescent signal is generated by the FAM dye when it detaches from the probe as the DNA polymerase in the SOLiD™ Library qPCR Mix extends the 3' strand.  - The uniformity of fragment size of libraries was confirmed with Bioanalyzer. For SOLiD fragment libraries the target size of the fragments is 150 - 300 bp.