RNA-SEQ of Melocactus glaucescens: in vitro shoot organogenesis
Total RNA and DynabeadsOligo were used to isolate mRNA. The resulting mRNA fragments of approximately 400 nucleotides were converted to double-stranded cDNA using random hexamer primers and corresponding enzymes, in accordance with standard DNA library protocols for sequencing. Briefly, cDNA was end-repaired, phosphorylated, and adenylated. Common TruSeq adapters containing 8 bp indexes suitable for Illumina sequencing were ligated to the adenylated molecules and the resulting libraries were amplified by 13 cycles of PCR to enrich for properly ligated molecules. The final libraries were quantified using PicoGreen and equimolarly combined into a single sample, which was then sequenced on an Illumina HiSeq 3000 instrument. Paired-end reads with an average length of 100 bp were obtained from two lines.