title: Control_3
design: The RNA of the total sample was isolated and purified by TRIzol (thermofisher, 15596018) according to the operation scheme provided by the manufacturer. Then the quantity and purity of total RNA was controlled by NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) and the integrity of RNA was detected by Bioanalyzer 2100 (Agilent, CA, USA). The concentration was >50ng/L, RIN value >7.0, and total RNA>1g. oligo(dT) magnetic beads (Dynabeads Oligo (dT), cat.25-61005, Thermo Fisher, USA) were used to specifically capture mRNA containing PolyA (polyadenylate) through two rounds of purification. The captured mRNA was fragmented using the NEBNextR RNA Fragmentation Module (cat.E6150S, USA) under high temperature conditions at 94 for 5-7 minutes. cDNA was synthesized from the fragmented RNA by means of Invitrogen SuperScriptTM II Reverse Transcriptase (cat.1896649, CA, USA). Then, E. coli DNA polymerase I (NEB, cat.m0209, USA) and RNase H (NEB, cat.m0297, USA) are used for double strand synthesis, and these complex double strands of DNA and RNA are converted into DNA double strands. At the same time, dUTP Solution (Thermo Fisher, cat.R0133, CA, USA) was added to the double-stranded DNA to complete the end of the double-stranded DNA to the flat end. Then an A base is added to each end of it, so that it can be connected with the terminal with T base joints, and the size of its fragment is screened and purified by magnetic beads. The two chains were digested with UDG enzyme (NEB, cat.m0280, MA, US), then kept at 95 for 3 minutes by PCR, denatured at 98 for a total of 8 cycles of 15 seconds each, annealed to 60 for 15 seconds, extended at 72 for 30 seconds, and finally extended at 72 for 5 minutes. To form a fragment size of 300bp50bp library (chain specific library). Finally, we used illumina NovaseqTM 6000 (LC Bio Technology CO.,Ltd. Hangzhou, China) to double-end sequencing in PE152 mode according to standard procedures.