Spatial host microbiome profiling of Homo sapeins : noninflamed and inflamed tissue of CD5
Based on the approach of McKellar et al., we conducted in situ polyadenylation.15 This procedure included equilibration with wash buffer containing Protector RNase Inhibitor (Roche, catalog no. 3335402001), followed by incubation with an enzyme mix containing yeast poly A polymerase (Thermo Scientific, catalog no. 74225Z25KU). We used the Visium Tissue Optimization Kit from 10x Genomics to determine the optimal tissue permeabilization time. After the in-situ polyadenylation step, we prepared final sequencing libraries using standard Visium library preparation protocol (CG000239, Rev F, 10x Genomics). The prepared libraries were combined and sequenced on a NovaSeq 6000 system (Illumina, San Diego, CA, USA) using the NovaSeq 6000 S1 Reagent Kit v1.5 (200 cycles, 20028318, Illumina). We sequenced to a depth of approximately 120M reads per sample.