GSM4792447: Control rep2; Saccharomyces cerevisiae; RNA-Seq
Extraction of total RNA was performed with RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. 1μg of total RNA was used for RNA-seq analysis.Libraries for RNA-Seq were prepared using the following procedure. At first, poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. After purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then had the addition of a single 'A' base and subsequent ligation of the adapter. The products were then purified and enriched with PCR amplification. Then the PCR yield was quantified by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. The DNBs were loaded into the patterned nanoarrays and pair-end reads of 100 bp were read through on the BGISEQ-500 platform for further data analysis. For this step, the BGISEQ-500 platform was combined the DNA nanoball-based nanoarrays and stepwise sequencing using Combinational Probe-Anchor Synthesis Sequencing Method. RNA libraries were prepared for sequencing using standard BGI protocols