title: RNA-Seq of mus musculus: adult male intestine
design: Total RNAwas extracted usingTrizol reagent (thermofisher, 15596018) following the manufacturer's procedure. The total RNAquantity and purity were analysis of Bioanalyzer 2100 and RNA6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511) , high-quality RNAsamples with RIN number > 7.0 were used to construct sequencing library.After total RNA was extracted, mRNAwas purified from total RNA(5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNAwas fragmented into short fragments using divalent cations under cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNApolymerase 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 containeda T-base overhang for ligating the adapter to theA-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed withAMPureXP 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 cDNAlibraryswere 30050 bp.At last, we performed the 2150bp paired-end sequencing (PE150) on an Illumina