H0h_2
Isolate and purify total RNA using TRIzol, and measure the quantity and purity of total RNA with NanoDrop. Validate RNA integrity with Bioanalyzer and agarose gel electrophoresis. Qualified RNA (concentration >50ng/L, RIN >7.0, OD260/280 >1.8, total amount >1g) is used for downstream experiments. Capture PolyA mRNA with oligo(dT) magnetic beads, fragment with NEBNext magnesium reagent kit, and synthesize cDNA with SuperScript II reverse transcriptase. Synthesize double-stranded DNA with E. coli DNA polymerase I and RNase H, incorporate dUTP, and ligate adapters with blunt ends. Purify with magnetic beads, digest with UDG enzyme, and amplify with PCR to form a library of 300bp50bp fragments. Finally, perform paired-end sequencing with Illumina Novaseq 6000.