GSM8695554: NoSucrose_R1; Saccharomyces cerevisiae; RNA-Seq
Cell pellets were re-suspended in TRIzol reagent supplemented with 5 mM DTT final,and lysed using a Fast Prep-24 machine (MP Biomedicals). RNA was cleaned twice with chloroform, treated with DNase I and purified using RNA Clean and Concentrator kit. Libraries were prepared from 500ng of each total-RNA. Libraries were prepared  using the NEBNEXT Ultra II Directional RNA Library Prep kit (NEB #7760) and the Poly-A mRNA magnetic isolation module (NEB #E7490).  Poly-A containing mRNA molecules were purified using poly-T oligo attached magnetic beads. Following purification the mRNA was fragmented using divalent cations under elevated temperature and primed with random hexamers. Primed RNA fragments were reverse transcribed into first strand cDNA using reverse transcriptase and random primers. RNA templates were removed and a replacement strand synthesised incorporating dUTP in place of dTTP to generate ds cDNA. The incorporation of dUTP in second strand synthesis quenches the second strand during amplification as the polymerase used in the assay is not incorporated past this nucleotide. AMPure XP beads (Beckman Coulter, #A63881) were then used to separate the ds cDNA from the second strand reaction mix, providing blunt-ended cDNA. A single 'A' nucleotide was added to the 3' ends of the blunt fragments to prevent them from ligating to another during the subsequent adapter ligation reaction, and a corresponding single 'T' nucleotide on the 3' end of the adapter provided a complementary overhang for ligating the adapter to the fragment. Adapter-ligated DNA was then purified with AMPure XP beads before 10 cycles of PCR with unique dual index sequences were used to selectively enrich DNA fragments with adapter sequences and allow multiplexed sequencing. After amplification libraries were purified using AMPure XP beads. Final libraries had an average fragment size of Xbp including index and adapter sequences.
GEO
GSM8695554_r1