Bulk RNA sequencing

Collect the ileum from a PBS-perfused mouse.

Thoroughly flush the ileum sample with cold 1x PBS.

Incubate samples in TRIzol reagent (Thermo Fisher Scientific, Cat #15596026; Waltham, MA) and store at -80°C until ready for bulk RNA sequencing.

Extract RNA and construct library using Illumina TruSeq chemistry.

Sequence the libraries using an Illumina NovaSeq6000.

Samples are multiplexed in each lane, which yielded targeted number of paired-end, 100bp reads for each sample. RTA (Illumina) for base calling and bcl2fastq2 (version 2.19) are used for converting BCL to fastq format, coupled with adaptor trimming.

Perform a pseudoalignment to a kallisto index created from transcriptomes (GRCm38) using kallisto (0.44.0).

Test differentially expressed genes under various conditions using DESeq2R packages designed to test differential expression between two experimental groups from RNA-seq counts data.

Genes are considered differentially expressed if they had an adjusted p-value <0.05 and a log2fold change below or above 0.5. Normalize the differential expression for each gene.