RNA-seq Library Preparation Protocol

Four separate libraries (CT, T_2, T_48 and RC) were sampled. Total RNA was extracted by TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. The quality of the RNA products was verified as described by Ren et al. (2014) [14 (link)]; RNase-free DNase I (Invitrogen, USA) was used to degrade any possible DNA, and oligo (dT)-coated magnetic beads were mixed with the total RNA to concentrate the ployA tailed mRNA. Fragmentation was performed by incubation in an NEB Next First Strand Synthesis Reaction Buffer (NEB, USA), and the second strand was generated with the buffer, dNTPs, RNase H and DNA polymerase-I. Adapters were ligated to the synthesized cDNA fragments after an end repair step.