RNA-seq of Camellia sinensis: leaf under shaded condition
Total RNA was extracted from the tea leaves using CTAB (Ambion, Austin, TX, USA) and then the RNA concentration and integrity were determined using the RNA 6000 Nano Reagents Part 1 kit and the 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The mRNA containing the poly-A tail was enriched using oligo(dT) magnetic beads. The rRNA was hybridized into a DNA probe, with the resulting DNA/RNA selectively digested first with RNaseH and then with DNaseI to eliminate the DNA probe. The desired RNA was obtained after a purification step. The obtained RNA was fragmented in a fragmentation buffer and then random hexamer primers were used for the reverse transcription to synthesize the first-strand cDNA sequence. After generating the second cDNA strand, the resulting double-stranded cDNA ends were modified to produce a blunt and phosphorylated 5 end and a 3 sticky end with a protruding A-tail. A bubble-like junction with a protruding T-tail was ligated to the 3 ends. The ligation products were amplified by PCR using specific primers. The PCR products were thermally denatured into single-stranded DNA and then a bridge primer was used to obtain a single-stranded circular DNA library, which was sequenced using the BGISEQ-500 sequencing platform.