RNA-seq Analysis of Mouse Colonic Tissue

RNA was extracted from snap-frozen colonic mouse tissue (Qiagen RNeasy mini kit cat # 74106, Venlo, The Netherlands) and RNA integrity and quantity was checked with Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). RNA sequencing and preparative techniques were performed by the Genomics Core (UZ Leuven, http://gc.uzleuven.be/). TruSeq stranded mRNA library preparation (Illumina, San Diego, CA, USA) was performed according to the manufacturer's guidelines. Sequencing was performed on the HiSeq2500 platform with a sequencing depth ranging from 9 to 21 M reads per sample (Illumina). The adaptors were trimmed from the reads with the use of ea_utils v1.1.2. Reads that were shorter than 25 bp after adaptor trimming were removed. The pre-processed reads were aligned to the reference genome of Mus musculus (Mm10) with TopHat v2.0.13. Counting of the reads was performed with HTSeq46 (link) or CuffQuant47 (link). Pathway analysis was performed with Ingenuity Pathway Analysis (IPA, Qiagen). Raw data have been deposited in BioProject with the accession code PRJNA374413 (https://www.ncbi.nlm.nih.gov/bioproject/374413).