GSM5068608: LCY001_GE_rep2; Saccharomyces cerevisiae; RNA-Seq
Yeast RNA was extracted using the TRIzol® method following the manufacturer's protocol. Bulk cells or filamentous fungus are grounded to a powder using liquid nitrogen, and the powder was transferred into the 2 mL tube contains 1.5 mL Trizol reagent. The mix was shaked for 3 min, and kept for 5 min at room temperature, then was Centrifuged at 10,000×g for 5 min at 4 °C. The supernatant was added 200 μL of chloroform/isoamyl alcohol (24:1) with 1 mL lysis reagents. After centrifuged at 10,000×g for 10 min 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 1 h. After centrifuged at 13,600×g for 20 min at 4 °C, the supernatant was precipitated by 1 mL of 75% ethanol and let dry for 3-5 min. The RNA pellet was dissolved with 30-100 μL of DEPC water or RNase-free water. The concentration of the extracted RNA samples 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). Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment 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. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).