RNASeq from colonic tissue of naïve IEC-Bmal1-/- mice and Bmal1flox controls over the course of a day

Circadian clocks are important for gut health. This experiment aimed to determine the role of core clock gene Bmal1. Mice were generated with Bmal1 selectively deleted in Villin-expressing cells (predominantly IECs). Colon tissue was harvested from Villin-Bmal1-/- and Bmal1flox controls over the course of the day at zeitgeber time (ZT)0, ZT6, ZT12 and ZT18. Colon tissue was homogenised in lysing MatrixD tubes (MP biomedical) containing TRIzol (Invitrogen), using a bead mill homogeniser (Fisherbrand). RNA was extracted with chloroform and aqueous phase was precipitated with isopropyl alcohol. Supernatant was washed with 70% ethanol and the RNA pellet was resuspended in nuclease-free water. Sequencing library preparation with TruSeq Stranded mRNA assay (Illumina) and sequencing were performed by the University of Manchester Genomic Technologies Core Facility. Multiplexed libraries were analysed by paired-end sequencing on NovaSeq 6000 (Illumina). Quality control was performed with Fastqc (v0.11.3) and FastqScreen (v0.14.0). Reads were trimmed using BBDuk from BBMap (v38.96) and reads were mapped to the mouse genome (mm39/vM30) using STAR (v2.7.10a).