title: Compartmentalized control of skin immunity by resident commensals
design: Ear and flank skin samples were sterilely obtained and processed using a protocol adapted from Grice et al (3). DNA was extracted from fecal pellets using QIAamp DNA stool mini kit (Qiagen). For quantitative analysis of 16S rDNA, real time PCR was performed using primers BacF (5’-CGGCAACGAGCGCAACCC-3’) and BacR (5’-CCATTGTAGCACGTGTGTAGCC-3’) (4). For sequencing of 16S rDNA amplicon libraries were prepared from sample DNA using Accuprime High Fidelity Taq polymerase (Invitrogen) and universal primers flanking variable regions V1 (primer 27F; 5’-AGAGTTTGATCCTGGCTCAG-3’) and V3 (primer 534R; 5’-ATTACCGCGGCTGCTGG-3’). For each sample, the universal primers were tagged with unique sequences ("barcodes") to allow for multiplexing/demultiplexing(5) PCR products were then purified using the Agencourt Ampure XP Kit (Beckman Counter Genomics) and quantitated using the QuantIT dsDNA High-Sensitivity Assay Kit (Invitrogen).
library_name: V1-V3 skin gut