Directional rna library prep kit

The RNA sequencing libraries were generated using the rRNA-depleted RNA by Directional RNA Library Prep Kit for Illumina (NEB, USA). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) Primer. The PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The detailed method was used as previously described [19 ].
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using HiSeq 2500 PE Cluster Kit (Illumina). After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and 125 bp paired-end reads were generated. The transcriptome sequencing was performed by NovelBio Corp. Laboratory, Shanghai, China. Raw data were firstly processed through in-house perl scripts to obtain clean data by removing reads containing adapter, ploy-N and with low quality that reads with >5% ambiguous bases (noted as N) and low-quality reads containing more than 20 percent of bases with qualities of <13. At the same time, Q20, Q30, and GC content of the clean data were calculated to meet the standard (Q20>90, Q30>85) [20 (link)].