RNASeq from colonic tissue of naive 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).昼夜节律钟（Circadian Clock）对肠道健康至关重要。本实验旨在明确核心时钟基因Bmal1的作用。本研究构建了在表达Villin的细胞（主要为肠上皮细胞，Intestinal Epithelial Cells，IECs）中特异性敲除Bmal1的小鼠模型。分别于授时因子时间（Zeitgeber Time，ZT）0、ZT6、ZT12及ZT18时刻，从Villin-Bmal1-/-敲除小鼠及其Bmal1flox对照小鼠中采集结肠组织。将结肠组织置于含TRIzol（Invitrogen）的裂解用MatrixD管（MP Biomedicals）中，使用珠磨式组织匀浆器（Fisherbrand）进行匀浆处理。采用氯仿萃取法提取RNA，收取水相后以异丙醇沉淀RNA。收集上清液，用70%乙醇洗涤RNA沉淀，随后将沉淀重悬于无核酸酶水中。采用Illumina TruSeq Stranded mRNA建库试剂盒完成测序文库构建，测序实验由曼彻斯特大学基因组技术核心设施承接完成。采用Illumina NovaSeq 6000测序平台对多重测序文库进行双端测序分析。使用Fastqc（v0.11.3）与FastqScreen（v0.14.0）对测序数据开展质量控制。利用BBMap（v38.96）内置的BBDuk工具对测序读段（Reads）进行剪切过滤，并通过STAR（v2.7.10a）将测序读段比对至小鼠基因组（mm39/vM30）。