3 mm glass beads

Total RNA was extracted from half a rectal mucosal biopsy using the miRNeasy Mini Kit (Qiagen, UK) following the manufacturer's instructions. Tissue disruption was performed by shaking the tissues with five 3 mm glass beads (VWR, UK) for 1 m in QIAzol Lysis Reagent (miRNeasy Mini Kit) using an amalgamator. The lysate and beads were transferred to QiaShredders (Qiagen, UK) for homogenization by centrifugation for 2 m at maximum speed. RNA was eluted in 30 µL of RNase‐free water. Concentration and RNA purity were determined using the NanoDrop 1000 spectrophotometer (Thermo Scientific) and NanoDrop 1000 Software version 3.7.1. RNA integrity was assessed by agarose gel electrophoresis.

Stool samples were thawed and re-suspended in phosphate-buffered saline (PBS; 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride) and homogenised in a stomacher for 2 min at 460 paddle beats per minute. The resulting faecal slurry was vortexed with 3-mm glass beads (VWR) for 30 s before being centrifuged at 400×g for 2 min at room temperature. The supernatant (375 μL) was fixed in 4% (w:v) (1125 μL) paraformaldehyde for 4 h at 4 °C. To wash the cells out of paraformaldehyde, samples were centrifuged at 13,000×g in 1 mL PBS for 5 min at room temperature; this centrifugation was repeated two more times, then samples were re-suspended in 150 μL PBS and stored in ethanol (1:1 by v:v) at − 20 °C for enumeration by fluorescence in situ hybridisation (FISH).2

Total RNA for mRNA analyses was extracted from half of a rectal mucosal biopsy by using the RNeasy Mini Kit (Qiagen) as described by the manufacturer. RNA including microRNA was extracted separately from half of a rectal mucosal biopsy by using the miRNeasy Mini Kit (Qiagen). Tissue disruption was performed by shaking the tissue samples with five 3-mm glass beads (VWR) for 1 min in Buffer RLT (RNeasy Mini Kit) or QIAzol Lysis Reagent (miRNeasy Mini Kit) by using an amalgamator. The lysate and beads were transferred to QiaShredders (Qiagen) for homogenization. RNA concentration and an indication of RNA purity were determined by using the NanoDrop 1000 spectrophotometer (Thermo Scientific) and NanoDrop 1000 Software version 3.7.1. RNA integrity was assessed by agarose gel electrophoresis.