Microbial RNA-seq analysis of MAG gene expression
For RNA extraction, approximately 50 mg of a faecal sample, collected from each participant at the baseline, 1-month and 3-month timepoints, was pulverized under liquid nitrogen with a mortar and pestle and aliquoted into 2 ml cryotubes. A 3.97 mm steel ball and 250 µl of 0.1 mm zirconia/silica beads were subsequently added to each sample tube, together with 500 µl of a mixture of phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.8–8.2), 210 µl of 20% SDS and 500 µl of 2× Qiagen buffer A (200 mM NaCl, 200 mM Trizma base, 20 mM EDTA). After a 1 min treatment in a bead beater (Biospec Minibeadbeater-96), the samples were centrifuged at 3,220As an initial pre-processing step, raw reads were aggregated by sample across the two NovaSeq runs, resulting in a total of 5.0 × 10Differential expression analysis (edgeRFrom these models, we identified genes that exhibited significant differential expression using the quasi-likelihood For subsequent functional metabolic pathway enrichment analyses, we (1) ordered transcripts assigned to WLZ-associated MAGs on the basis of a ranking metric calculated as the direction of the fold-change × −logFor targeted transcriptional analyses of the CAZymes encoded by 