Supplementary titles and legends to figures
Sup. Figure 1. Summary of the experimental design in this study. Three independent samples sets were used to obtain microbial populations from the interfold and digesta regions by laser capture microdissection (LCM). Microbial populations were characterized by 16S rRNA gene analyses using tag pyrosequencing, Sanger sequencing, quantitative PCR and fingerprinting methods. WT = adult (8-12 week old) wild-type C57/Bl6 mice. Bacteroidaceae* = primers also amplifies 16S rRNA genes of
Porphyromonadaceae and Prevotellaceae. Known type strains
¥
= known type strains were identified and retrieved from RDP using Seqmatch search.
Sup. Figure 2. Dense fusiform-shape bacteria located between the transverse folds of the mouse ascending colon.
(A) A section of a transverse fold from the murine ascending colon stained with
Bisbenzimide (Hoechst 33342) to label DNA blue. Dense microbial populations are labeled in the interfold region (denoted by white dashed lines). The section highlighted by the red box is shown in panel
B. (B) Higher power view of interfold region of microbial populations localized in the interfold region shows that the staining is consistent with microbial nuclei. Bars = 50 μm (A) and 5 μm (B).
Sup. Figure 3. Group-specific primer sets are highly sensitive, quantitative and linear over a wide range of target DNA concentrations for both Gram-positive and Gram-negative microbes.
(A) DNA was isolated from bacterial type strains representing each of the groups targeted by group-specific primers. a (BF), Bacteroides fragilis ; (CP), Clostridium perfringes ; (EC), Escherichia coli ; (EF),
Enterococcus faecium ; (LA), Lactobacillus acidophilus ; (RP), Ruminococcus productus . b Bacteria universal primer set, Bact-8F and Bact-1391R. c Bacteroidaceae-Porphyromonadaceae-Prevotellaceae group. (B) DNA concentration was measured by spectrophotometry and a range of known concentrations from each strain was amplified by quantitative PCR using corresponding primer sets. All primer sets demonstrate a linear relationship for DNA concentrations from 100 fg to 10 ng, with some sets showing a

lower limit of detection ≤ 10 fg.
(C) Gram-positive and Gram-negative bacterial DNA was efficiently extracted from simple mixtures over a large range of sample material. Liquid cultures of Escherichia coli and Enterococcus faecalis type strains were normalized by optical density, mixed at defined ratios, and fixed on slides. Genomic DNA was extracted and used as DNA template for quantitative PCR assays using group-specific primers. Plots of cycle threshold (Ct) values for both type strains in defined ratios are shown (n = 3 experiments). Dashed line = hypothetical curve for unbiased extraction and 100% PCR efficiency.
Sup. Figure 4. Pyrosequencing rarefaction curves depicting the number of Operational Taxonomic
Units identified at different levels.
Rarefaction curves at 97% (blue line), 95% (red line), 90% (green line) and 80% (purple line) confidence interval. Each chart represents an intestinal region. Sequences were obtained from pooled samples (n = 3) of interfolds (29,560 reads) and digesta (38,120 reads) regions. Reads were clustered into Operational Taxonomic Units (OTUs) at different pairwise distances using the complete-linkage clustering algorithm at the RDP.