control 2
mRNA was enriched by binding to the oligo (dT)-attached magnetic beads and fragmented into small pieces with the fragment buffer at appropriate temperature. Afterwards, random N6 primers were used in the reverse-transcription to generate the first-strand cDNA, followed by a second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and then was 3 adenylated. Adapters were ligated to the ends of these 3 adenylated cDNA fragments. The cDNA fragments were amplified by PCR and further validated for quality control. The PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. DNA nanoballs (DNBs), which amplify from final library, were loaded into the patterned nanoarray. And single end 50 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).