RNA-seq
Total RNA was extracted from tissues using TRIzol (Invitrogen, Carlsbad, California, USA) according to manual instruction.  Appropriate amount of tissue was ground into powder under liquid nitrogen, then transferred into a 2ml EP tube containing 1.5ml of TRIzol, standing for 5 minutes. For cell samples, the TRIzol was directly added to the cell sample with the amount 1 mL per 110^6 cells, followed by vortex mixing and standing for 5-minute. The mixture was centrifuged at 4C at 12,000xg for 5 minutes, and the supernatant was transferred to a new EP tube for extraction. 300l of chloroform/isoamyl alcohol (24:1) was added into the transferred supernatant and the mixture was vigorously vortexed to ensure thorough mixing. Then the mixture was centrifuged at 4C and 12,000xg for 8 minutes. Take the clear upper aqueous layer and repeat the above process one time. After centrifugation, the final upper aqueous layer was carefully transferred to a new 1.5ml EP tube with a 2/3 supernatant volume of isopropanol (for low amount of tissues/ cell, 2L 5mg/mL glycogen can be added for precipitation), gently inverted to mix well, placed in a -20C refrigerator for 2 hours. Next, the precipitation mixture was centrifuged with 17500xg at 4C for 25 minutes. The supernatant was discarded, and the precipitation was washed with 0.9ml of 75% ethanol. The precipitation was suspended by inverting the tube up and down several times. Then the precipitation was collected by centrifuging at 17500g for 3 minutes at 4C and discarding the supernatant. Repeat the centrifugation to remove supernatant completely and the precipitation was dried in the biosafety cabinet for 3-5 minutes. Finally, 20L~200L of DEPC-treated or RNase-free water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a Fragment Analyzer or Agilent 2100 Bioanalyzer (Agilent, CA, USA), or Qseq-400 (Bioptic, Taiwan, China).