S. oleracea opt. TruSeq rep2
Leaves of S. oleracea were disrupted and mixed with Trizol. Total RNA and 3'-adapter (5'-pUGGAATTCTCGGGTGCCAAGG-amino-C7-3') were ligated. The SuperScript IV RT were added. The cDNA was separated on a poly-acrylamide gel. The DNA-only version of the Illumina TruSeq small RNA kit adapter (5'-pGATCGTCGGACTGTAGAACTCTGAAC-AminoC6-3') was ligated to the gel-purified cDNA. cDNA carrying 5' and 3' adapter sequences was incubated and amplified with the Illumina 5' PCR primer for TruSeq small RNA (5'- AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3') and a custom index primer (5'-CAAGCAGAAGACGGCATACGAGATNNNNNNTAGGACTCCATGCAAGCTCGGTACCGACAGTG-3'). High-throughput analysis of the libraries was done as single end run (150 nt) on a Illumina MiSeq System with the custom primer (5'-CACTGTCGGTACCGAGCTTGCATGGAGTCCTA-3').