RNA-Seq of Rattus norvegicus:RCS
Total RNA was extracted using Trizol reagent (thermofisher, 15596018) following the manufacturer's procedure. The total  RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA,  5067-1511) , high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library. After total RNA  was extracted, mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two  rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under  elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94 5-7min). Then the  cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript II Reverse Transcriptase (Invitrogen,  cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I  (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An  A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter  contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to  the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB,  cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the  following conditions: initial denaturation at 95 for 3 min; 8 cycles of denaturation at 98 for 15 sec, annealing at 60  for 15 sec, and extension at 72 for 30 sec; and then final extension at 72 for 5 min. The average insert size for the final  cDNA librarys were 30050 bp. At last, we performed the 2150bp paired-end sequencing (PE150) on an Illumina  Novaseq 6002 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.