RNA-Seq of Prunella Spica
A total of 1 g RNA per sample is used as input material for RNA sample preparation. The NEBNext Ultra RNA Library Preparation Kit for Illumina (NEB, USA) was used to generate the sequencing library, and the index code was added to the attribute sequence of each sample. Use magnetic beads with poly-T oligonucleotides to purify mRNA from total RNA. At high temperature, the divalent cations are cleaved in NEBNext first-strand synthesis reaction buffer (5X). The first strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase. Subsequently, DNA polymerase I and RNase H were used for second-strand cDNA synthesis. The remaining overhangs are converted to blunt ends by exonuclease/polymerase activity. After the 3'end of the DNA fragment is adenylated, the NEBNext adaptor with a hairpin loop structure is ligated for hybridization. In order to select cDNA fragments preferably 240 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA). Then use 3 L of USER enzyme (NEB, USA) with the size-selected, adaptor-linked cDNA at 37 C for 15 min, then at 95 C for 5 min, and then perform PCR. Then use Phusion high-fidelity DNA polymerase, Universal PCR primers and Index (X) Primer for PCR. Finally, the PCR product was purified using the AMPure XP system (Beckman Coulter, Beverly), and the library quality was evaluated by the Agilent Bioanalyzer 2100 (Agilent, USA). The TruSeq PE Cluster Kit v3-cBot-HS kit (Illumina, USA) was used to cluster the index-encoded samples on the cBot cluster generation system. After the clusters were generated, the library preparations were sequenced by the Illumina HiSeq 2000 platform (Illumina, USA), and paired-end reads were generated.
Prunella Spica