title: GSM8440221: WT_3; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: Cells were homogenized by mechanical disruption in 1.5 mL TRIzol lysis buffer. Samples were centrifuged at 12,000g for 5 min in 4ºC. The supernatant was transferred to new centrifuge tubes containing a chloroform/isoamyl alcohol (24:1) mix. After each shaking step, the samples were centrifuged at 12,000g for 8 minutes at 4ºC. Then, the supernatant was transferred to a new centrifuge tube containing isopropyl alcohol (2/3 vol/vol), gently shaken and cooled at -20ºC. Samples were then centrifuged for 25 minutes at 17,500 g (4ºC) and the supernatant was transferred to new microcentrifuge tubes, and combined twice with 75% (vol/vol) ethanol and mixed by pipetting. After centrifugation at 17,500 g for 3 minutes (4ºC), the supernantant was discarded, and the pellet was resuspended in 20-200 μL DEPC-H2O or RNase-free water. mRNA enrichment was performed on total RNA using oligo(dT)-attached magnetic beads. The enriched mRNA with poly(A) tails was fragmented using a fragmentation buffer, followed by reverse transcription using random N6 primers to synthesize cDNA double strands. The synthesized double-stranded DNA was then end-repaired and 5'-phosphorylated, with a protruding 'A' at the 3' end forming a blunt end, followed by ligation of a bubble-shaped adapter with a protruding 'T' at the 3'end. The ligation products were PCR amplified using specific primers. The PCR products were denatured to single strands, and then single-stranded circular DNA libraries were generated using a bridged primer. The constructed libraries were quality-checked and sequenced after passing the quality control.