title: GSM5516142: Batch2 Saikosaponin C 2uM rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated from tissue using Maxwell (Promega) based method. One 1 mg of total RNA was processed for preparing mRNA sequencing library using MGIEasy RNA Directional Library Prep Kit (MGI) according to manufacturer's instruction. The first step involves purifying the poly-A containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the mRNA 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 QauntiFluor ONE dsDNA System (Promega). The library is circularized at 37 °C for 30 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 QauntiFluor ssDNA System (Promega). Sequencing of the prepared DNB was conducted on the MGIseq system (MGI) with 100 bp paired-end reads.