GSM3937644: Col-0 rep3; Arabidopsis thaliana; miRNA-Seq
Arabidopsis seedlings were collected, frozen in liquid nitrogen, and RNA was harvested using Trizol reagent. Total RNAs from 14-day-old seedlings were resolved on a 15% denaturing polyacrylamide gel and RNAs of 40-400 nt were recovered from the gel. The size-fractionated RNAs were ligated to a 3' adaptor using T4 RNA ligase 2, truncated (NEB). The 3' adaptor-ligated RNAs were resolved on a 15% denature polyacrylamide gel and RNAs of 60-420 nt were recovered. The size-fractionated RNAs were reverse-transcribed with an RT primer that is complementary to the 3' adaptor using SuperScript III (Life Technologies), followed by two-step PCR amplification. The cDNA was first amplified with the RT primer and a mixture of pre-miRNA-specific forward primers for 10 cycles and secondly amplified for 16 cycles with the 2nd PCR forward and reverse primers using Phusion DNA polymerase (NEB).  The PCR products were resolved on a 12% native polyacrylamide gel, and DNAs of 150-230 bp were recovered from the gel as the pre-miRNA 3' RACE-seq library. The library was sequenced on Illumina HiSeq3000.
GEO Accession
GSM3937644