GSM5503450: VEH/SIV_LB49; Macaca mulatta; RNA-Seq
Total RNA was extracted from gingival tissues at necropsy using the miRNeasy kit (Qiagen Inc). Transcriptome profiling by RNA-seq and data analysis were performed by Novogene (Sacremento, CA). cDNA library construction and sequencing were performed by Novogene Co. Ltd, Beijing, CA (http://www.novogene.cn/). For library construction, ~3 μg of total RNA from each sample was used to enrich mRNA with the poly-T oligo-attached magnetic beads. The purified mRNA was then randomly cleaved into small fragments using NEBNEXT RNA fragmentation buffer following the manufacturer's instructions. First-strand cDNA was synthesized using random hexamers and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA synthesis by nick translation was subsequently performed using E coli DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. The final cDNA library was prepared after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment. The cDNA fragments of preferentially 250−300 bp in length were selected using with AMPure XP system (Beckman Coulter, Beverly, U.S.A.) and PCR amplified using Phusion High-Fidelity DNA polymerase (NEB, Beijing, China), Universal PCR primers and Index (X) Primer. PCR products were then purified using AMPure XP system (Beckman Coulter, U.S.A.), and library quality was assessed on the Agilent Bioanalyzer 2100 system (Agilent Technologies, U.S.A.). The clustering of the index-coded samples was performed on a cBot Cluster Generation System, using TruSeq SR Cluster Kit v3-cBot-HS (Illumina), according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq 6000 platform and 150 bp paired end reads were generated.