title: control
design: totalRNA was treated by mRNA enrichment method or rRNA removal method, and mRNA enrichment: mRNA enrichment with polyA tail was performed by OligodT magnetic beads;RRNA removal: DNA probe hybridization rRNA and RNaseH selective digestion of DNA/RNA hybridization strand, followed by DNaseI digestion of DNA probe, and the required RNA was obtained after purification.Fragment the RNA obtained by using the interrupt buffer, reverse transcribe the random N6 primers, and then synthesize the two strands of cDNA to form the double-stranded DNA. The synthetic double-stranded DNA ends are flattened and phosphorylated at the 5 'end, with the 3' end forming A sticky end that protruded an' A ', and then connected to A bubbling junction with A 'T' protruding from the 3' end. The ligands were amplified by PCR using specific primers. The PCR product was thermally denatured into a single strand, and then a bridge primer was used to cyclize the single strand DNA to obtain a single strand circular DNA library. On-machine sequencing.