LM2 (CRX2080415)
RNA was extracted from the yeast cells using the RNAprep Pure Total RNA Extraction Kit (Tiangen, DP430). RNA library preparation and quality control were performed by Novogene (Beijing). Briefly, the preparation involves using total RNA as the starting material. mRNA with polyA tails is enriched using Oligo(dT) magnetic beads. The enriched mRNA is then fragmented into smaller pieces using divalent cations in a fragmentation buffer. The fragmented mRNA serves as a template for the first strand of cDNA synthesis, which is performed using random oligonucleotide primers and the M-MuLV reverse transcriptase system. After the RNA strand is degraded by RNase H, the second strand of cDNA is synthesized in a DNA polymerase I system using dNTPs. The resulting double-stranded cDNA is subjected to end repair, A-tail addition, and adapter ligation for sequencing. cDNA fragments of approximately 370-420 bp are selected using AMPure XP beads, then amplified by PCR and purified again with AMPure XP beads to generate the final library. Following library quality control, different libraries are pooled according to their effective concentration and target sequencing requirements. The pooled libraries are then sequenced using Illumina. The image data generated by the high-throughput sequencers are converted into sequence data (reads) via CASAVA base calling, producing files in fastq format. Raw data is then filtered for further analysis.