title: RNA-Seq of mus musculus: adult male liver
design: RNase H was used to remove the rRNA, DNase I was used to digest double-stranded and singlestranded DNA in total RNA. Purified RNA from previous steps was fragmented into small pieces with fragment buffer at appropriate temperature. Then, First-strand cDNA was generated in First Strand Master Mix by PCR, and the second-strand cDNA was generated as well. The reaction product was purified by magnetic beads, afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments were amplified with adapters by PCR, and the products was purified by Ampure XP Beads. Library was validating on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR product from previous step was heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29(Thermo Fisher Scientific, MA, USA) to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on BGISEQ500 platform (BGI-Shenzhen, China).