Polyatract mrna isolation systems kit

Total RNA was isolated separately from the 30 samples using a TRIzol Kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. The construction of a cDNA library and transcriptome sequencing was conducted in Novogene (Beijing, China), using the PolyATract mRNA Isolation Systems Kit (Promega) and Illumina HiSeq 4000 according to the manufacturer’s instructions.

Total RNA was isolated separately from the 45 samples using a TRIzol Kit (Invitrogen, CA, USA) according to the manufacturer's instructions. The RNA integrity was checked using an Agilent 2100 BioAnalyzer (Agilent Technologies, CA, USA), and the mRNA was purified from the total RNA using the PolyATract mRNA Isolation Systems Kit (Promega) following the manufacturer's instructions. Fragmentation was undertaken using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer. First stand cDNA was synthesized using a random hexamer primer and M-MuLV reverse transcriptase (RNase H-). The second strand cDNA synthesis was then performed using DNA polymerase I and RNase H. In order to select cDNA fragments that were preferentially 150–200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). The adaptor modified fragments were selected by gel purification and amplified through PCR to create the final cDNA library. Transcriptome sequencing was conducted using Illumina NovaSeq 6000 according to the manufacturer's instructions.