GSM1421974: CT Inf. Meristem 14 [small RNA-seq]; Brassica rapa; miRNA-Seq
All tissue samples harvested from control and treated plants were subjected to total RNA isolation using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Two independent RNA samples per every tissue were prepared. Total RNA was further purified and concentrated using the RNA Clean-Up and Concentration Kit (Norgen Biotek Corp., Ontario, Canada). The quality and concentration of every sample were quantified using the NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific Inc.) and the absorption ratios at 260/280 and 260/230 nm wavelengths. Agarose gel electrophoresis was performed to verify RNA sample integrity. Small non-coding RNA libraries were generated from 16 samples of total RNA in 2 biological replicates for most of the tissues using the TruSeq small RNA library construction kit according to the manufacturer’s protocol (Illumina, San Diego, CA, U.S.A.). Briefly, the 3΄ and 5΄ adapters were ligated to small RNAs from the total RNA sample followed by reverse-transcription PCR amplification. PCR was performed with two primers that annealed to the ends of adapters and contained indexes. Subsequently, the libraries with unique indexes were pooled together; the cDNA was gel-purified using a TBE PAGE gel and then concentrated by ethanol. Following a successful library quality control by qPCR, flow cell cluster generation was performed using a cBot. Single-end multiplexed sequencing was done using the Illumina GAIIx platform with the total of 36 cycles.