Perfecta sybr mix

RNA was extracted using an OMNI Bead Ruptor and 2.8 mm ceramic beads (OMNI International) in RLT buffer followed by Qiashredder and RNeasy kit using Qiacube (Qiagen) automated extraction according to manufacturer’s specifications. Total RNA was quantified using Quant-iT RNA Assay Kit (Thermo Fisher Scientific). Complementary DNA was prepared using qScript reverse transcription kit (Quantabio) and qPCR was performed using Perfecta SYBR mix (Quantabio) and StepOne Plus Real Time PCR system and software (Applied Biosystems). RNA libraries were prepared with QuantSeq 3’mRNA-Seq Library Prep Kit (Lexogen) and sequenced using Illumina NextSeq. Sequences were processed to remove adapter, polyA and low-quality bases by BBTools (https://jgi.doe.gov/data-and-tools/bbtools/) using bbduk parameters of k = 13, ktrim = r, forcetrimleft = 12, useshortkmers = t, mink = 5, qtrim = r, trimq = 15, minlength = 20.
Reads were aligned to mouse genome and transcriptome (ENSEMBL NCBIM37) using Tophat (v2.1.1) ^{77 (link)}with default parameters. Number of reads per million for mouse genes were counted using HTSeq (v 0.6.0)^{78 (link)} and quantile normalized. BRB-ArrayTools was used to identify genes differentially expressed in the liver and ileum when supplemented with or without the Lactobacillus candidates. Pathway enrichment was performed using Metascape^{79 (link)}.