GSM7611127: S2N; Rattus norvegicus; miRNA-Seq
Total RNA was isolated using the miRvana PARIS kit (AM1556) from Invitrogen according to the manufacturer's protocol. Small-RNA sequencing was performed on four biological replicates per group by BGI Genomics (BGI Americas Corp, Cambridge, MA, USA). Total RNA was assessed for quality before library preparation and RNA Integrity Numbers (RIN) were ≥ 7.5 with 28S/18S ≥ 1.3. Small RNAs (18-30 nt) were separated from total RNA by PAGE gel. Selected small RNAs were ligated with 3' and 5' adapters. The formed strands were reverse transcribed in cDNA and PCR amplified with high-ping polymerase. The PCR products (100-120bp) were separated by PAGE gel to eliminate primer-dimers and other byproducts and purified. The PCR yield was quantified followed by single strand DNA cyclization (ssDNA circle) for final library construction. DNA nanoballs (DNBs) were generated by ssDNA circle by rolling circle replication. The DNBs were then loaded onto the BGISEQ-500 platform sequencing for 100bp paired end reads.