BTZ2 (CRX2062721; CRA031287)
The library was prepared following a standard workflow for transcriptome sequencing. Briefly, mRNA was enriched from total RNA using oligo(dT) magnetic beads and then fragmented into short pieces of approximately 300 bp using a fragmentation buffer. Using these mRNA fragments as templates, first-strand cDNA was synthesized with random hexamer primers. Subsequently, second-strand cDNA was synthesized using the first strand as a template by adding buffer, dNTPs, RNase H, and DNA polymerase I. The double-stranded cDNA was purified using AMPure XP beads. The purified cDNA then underwent end repair, poly-A tailing, and sequencing adapter ligation. Following this, AMPure XP beads were used to select the appropriate fragment size. The library was then amplified via PCR to generate the final cDNA library. Quality control and quantification were performed using methods such as Q-PCR to ensure an effective library concentration greater than 3 nM. Finally, different libraries were pooled according to the target data volume for sequencing on an Illumina platform.