12 days growth of R.salina
Total RNA was extracted from the fresh R.salina cells. mRNA is enriched by oligo (dT) -attached magnetic beads from the denatured RNA sample with the opened secondary structure. Then the mRNAs are fragmented and converted to double-stranded cDNA fragments, which then are subjected to end-repair and adaptor ligation. The cDNAs were amplified by PCR to construct cDNA library. The reaction system and program for circularization of products are subsequently configured and set up. Single-stranded cyclized products are produced through denaturing the PCR products and circularization reaction, while uncyclized linear DNA molecules are digested. Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS).