DNBSEQ-G400 paired Sequencing of T5 (KAS24170782) (KAE24107156)
The 500ng of total RNA was processed for preparing whole transcriptome sequencing library. Enrichment of whole transcriptome RNA by depleting ribosomal RNA (rRNA) was processed using MGIEasy RNA Directional Library Prep Kit (MGI) according to manufacturers instruction. After the rRNA is depleted, the remaining RNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved in the RT directional buffer, followed by second strand cDNA synthesis. These cDNA fragments then have the addition of a single A base and subsequent ligation of the adapter. The products are then purified and enriched with PCR to create the final cDNA library. The double stranded library is quantified using QuantiFluor ONE dsDNA System (Promega). The library is circularized at 37 C for 60 min, and then digested at 37 C for 30 min, followed by cleanup of circularization product. To make DNA nanoball (DNB), the library is incubated at 30 C for 25 min using DNB enzyme. Finally, Library was quantified by QuantiFluor ssDNA System (Promega). Sequencing of the prepared DNB was conducted on the DNBSEQ platform (MGI) with 150 bp paired-end reads.