GSM7823382
Total RNA was isolated using TRIzol® (Life Technologies) according to the manufacturer's instructions with the addition of a DNase treatment and overnight precipitation. Samples were resuspended in 12 µl RNase-free water and frozen at -80°C until further use. Total RNA samples were next depleted of both cytoplasmic and mitochondrial rRNA using Ribo Zero Gold (TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold; Illumina) and then fragmented using divalent cations under elevated temperatures.  Next generation RNA-seq was conducted using HiSeq® SBS Kit v4 (FC-401-4002), HiSeq® SR Cluster Kit v4 – cBotTM (GD-401-4001) and TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold Set B (RS-122-2302). The products were then purified using RNAClean XP Beads.  First strand cDNA was generated using SuperScript II Reverse Transcript (Thermo Fisher Scientific).  Second strand synthesis was achieved using DNA Polymerase I and RNaseH and the products were purified using AMPure XP Beads. The double stranded cDNAs were prepared for ligation of the multiple indexing adapters by adenylating the 3' blunt ends of the fragments. One adenine (A) nucleotide was added to the blunt fragments to prevent them from ligating to each other during the adapter ligation reaction. One corresponding thymine (T) nucleotide present on the 3ʹ end of each of the indexing adapters provides a complementary overhang for ligating to the fragment. This strategy ensures a low rate of chimera (concatenated template) formation.  The fragments were then purified using a 2 stage AmpPure XP bead cleanup.  Only fragments with an adapter on both ends can both bind to the surface-bound primers on a flowcell and form clusters. To enrich the DNA for these fragments, and to amplify the amount of DNA in the library, 15 cycles of PCR were used followed by an additional AmPureXP bead cleanup.  All 60 libraries were pooled and then clustered using an eight-lane high density flowcell (HiSeq® SR Cluster Kit v4 – cBotTM), on an Illumina cBot-2.  These clustered flow cells were then sequenced on an Illumina HiSeq 2500 DNA sequencer for 141 cycles with an additional 8 sequencing cycles to identify the sample indexes (HiSeq® SBS Kit v4). The libraries were sequenced on Illumina HiSeq 2500 and the images were processed with the corresponding Illumina RTA software version v1.18.66.3 and base-calling was performed using bcl2fastq v2.18.0.12.  Libraries were generated with single-end sequencing.