The heat treatment of 40 centigrade 6h
Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Secondly, the mRNA was broken into short fragments and cDNAwas synthesized using random hexamer primer, M-MuLV Reverse Transcriptase (RNaseH-Clustering and sequencing (Novogene Experimental Department)-), DNA Polymerase I and RNase H. Thirdly, blunt ends were created in the cDNAfragments which then were ligated to NEBNext adapters. Finally, five libraries with an insert sizeof 150-200 bp were constructedand then sequenced using an IlluminaHiseq 2500 platform.