DNBSEQ-T7 paired end sequencing of SAMD01608679
After purifying mRNA using oligo(dT) beads (BGI, LB00V60), the mRNA was fragmented, and cDNA was synthesized using the Optimal Dual-mode mRNA Library Prep Kit (BGI, LR00R96). The resulting double-stranded cDNA fragments were subjected to end repair, followed by the addition of a single 'A' nucleotide to the 3' ends of the blunt fragments. Adapters were ligated to the fragment ends using the BGI Plug-In Adapter Kit (BGI, LA00R04), and the cDNA was amplified by PCR. The PCR products were heat-denatured to obtain single-stranded DNA, which was circularized using the MGIEasy Circularization Kit (BGI, 1000005259). 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).