GSM1986158: 24#-12 RNA-seq; Gossypium hirsutum; RNA-Seq
Total RNA was extracted according to a guanidine thiocyanate method The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. These products are then purified and enriched with PCR to create the final cDNA library.  The cDNA library of each sample was constructed according to the instructions given in “Preparing Samples for Sequencing of mRNA Test Kits” (Illumina). Products were checked to verify the cDNA library quality and fragment length using an Agilent 2100 DNA 1000 Kit. Sequencing of the resulting cDNA library was carried out on an Illumina HiSeq 2000 paired-end 100-bp (PE 100) system.