DNBSEQ-T7 paired end sequencing; Transcriptome analysis of Saccharomyces cerevisiae petites
Transcriptome analysis of Saccharomyces cerevisiae petites
Cells were grown in YPD broth at 30˚C from OD600=0.01 till OD600=1.0. Pellets of 5ml cell culture were obtained by centrifuging at maximum speed for 1min. the pellets were fast frozen in liquid nitrogen. RNA was extracted using the TRIzol method following the manufacturer's protocol. Cell pellets were transferred from liquid nitrogen into the 2ml tub containing 1.5ml Trizol reagent. The mix was shared for 3min, and kept for 5in at room temperature, then centrifuged at 10,000xg for 5min at 4˚C. The supernatant was added 200µl of chloroform/isoamyl alcohol (24:1) with 1ml lysis reagents. After centrifuged at 10,000g for 10min at 4˚C, the supernatant was transferred into another new tube with equal volume of isopropanol and put in the refrigerator at -20˚C for 1h. After centrifuged at 13600xg for 20min at 4˚C, the supernatant was precipitated by 1ml of 75% ethanol and let dry for 3-5min. The RNA pellet was dissolved with 30-100µl of DEPC water. The concentration of the extracted RNA was determined using a Nanodrop system (NanoDrop, Madison, USA), and the integrity of the RNA was examined by the RNA integrity number (RIN) using an Agilent 2100 bioanalyzer (Agilent, Santa Clara, USA) buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library.