1. Transfer appropriate sample to corresponding grinding tube, then add 1.5 mL TRIzol lysis buffer, transfer to the grinding machine, grind 30 s, stay at 5 min. Dispense 1.5 mL of TRIzol lysis buffer to a 2 mL tube, and take appropriate amount of tissue samples, grind the tissue samples into powder with liquid nitrogen and transfer into the lysis buffer.  Stand flat for 5 min for the fully lysis of tissue cells. 2. Centrifuge the tissue samples after grinding and crushing at 12000×g for 5 min in 4℃. Transfer the supernatant to the centrifuge tube with 300 µL Chloroform/isoamyl alcohol (24:1) mix thoroughly by upside down violent shaking；Centrifuge at 12000×g for 8 min in 4℃; Transfer the supernatant to the 1.5 mL centrifuge tube, add 2/3 volume of Isopropyl alcohol, Gently invert and mix, place in -20℃ refrigerator for more than 2 h. Centrifuge at 17500×g for 25 min in 4℃. Discard the supernatant, wash with 0.9 mL 75% ethanol, suspend the precipitation by inverting. Centrifuge at 17500×g for 3 min in 4℃;Discard the supernatant, centrifuge for a short time and use the tip to remove the residual liquid, air dry for 3-5 min.Dissolve the precipitation with 20-200 μL DEPC-H2O or RNase-free water. 1 Sample QC; 2 mRNA is enriched by oligo (dT) -attached magnetic beads; 3 mRNA Fragmentation; 4 cDNA Synthesis; 5 End Repair, Add A and Adaptor Ligation; 6 PCR; 7 Library QC; 8 Circularization and make DNB; 9 Sequencing on DNBSEQ platform