Baseclear

16S sequencing of the V3‐V4 region was performed by BaseClear (Leiden, The Netherlands) and the results were analyzed using the CLC bio genomics workbench (Qiagen, The Netherlands), Microbial Genomics Toolbox. The SILVA 16S v128 99% database was used as reference database. Results show the relative abundances at phylum level for the three different donors.

Spores from mutant strain mcwc-1a∆ and the wild type R7B were confronted with a mouse macrophage cell-line (J774A.1 or ATCC TIB-67) in a 1.5:1 ratio as described in previous studies to perform host–pathogen interaction assays [12 (link)]. Briefly, the confrontation occurred in L15 medium supplemented with 20% fetal bovine serum (both from Capricorn Scientific) at a temperature of 37 °C for 5 h to ensure all spores were phagocytosed. As a non-confrontation control or saprotrophic conditions, the same concentration of spores was cultured in L15 medium supplemented with fetal bovine serum. Samples were prepared in quadruplicate.
Duplicates of each sample were pooled together, and RNA was extracted using the standard procedure of the RNeasy plant minikit (Qiagen, Hilden, Germany), resulting in two replicated RNA extractions per sample that were sent for sequencing to BaseClear (Leiden, The Netherlands). There, mRNA was enriched by poly(A) purification capture, and cDNA libraries were prepared using TruSeq RNA library preparation kits. The cDNA library was sequenced using an Illumina HiSeq 2500 system, producing two replicated raw datasets of first-stranded (or reverse), 50 bp, single-end reads.