RNA-SEQ of SCLC derived from H1
mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads. mRNA molecules were fragmented into small pieces using fragmentation reagent after reaction a certain period in proper temperature. Add an appropriate amount of primers to the interrupted sample, mix well, and react at a suitable temperature on a thermal cycler for a certain period of time to open the secondary structure and combine with the primers. Then add the first-strand synthesis reaction system Mix prepared in advance. Synthesize first-strand cDNA according to the corresponding procedure on the instrument. Prepare a second-strand synthesis reaction system (using dUTP instead of dTTP). React on Thermomixer at an appropriate temperature for a certain time to synthesize second-strand cDNA. The reaction product was purified by magnetic beads. Prepare the end repair & add "A" reaction system, react on a thermal cycler for a certain time, under the action of enzymes, repair the sticky ends of the cDNA double-stranded by reverse PCR products were purified with XP Beads, and dissolved in EB solution. Library was validated on the Agilent Technologies 2100 bioanalyzer. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library.The DNBs were load into the patterned nanoarray and single end 50 (pair end 100/150) bases reads were generated in the way of combinatorial Probe-Anchor Synthesis (cPAS).