FH8W
Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmentedinto small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA wasgenerated using random hexamer-primed reverse transcription, followed by a second-strand cDNAsynthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to endrepair. The cDNA fragments obtained from previous step were amplified by PCR, and productswere purified by Ampure XP Beads, then dissolved in EB solution. The product was validated onthe Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR productsfrom previous step were heated denatured and circularized by the splint oligo sequence to get thefinal library. The single strand circle DNA (ssCir DNA) was formatted as the final library. Thefinal library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 basesreads were generated on BGIseq500 platform (BGI-Shenzhen, China).