title: GSM8979621: S92 (53B_38b); Mus musculus; OTHER
library_construction_protocol: We extracted total bacterial DNA with QiaAMP Fast DNA stool Mini kit (Qiagen # 51604) according to the manufacturer's instructions. In the final step DNA was eluted in 80ul AE elution buffer. Amplicons targeting the V4 region of the bacterial 16S rRNA gene were generated using KOD One PCR Master Mix (Sigma-Aldrich) and universal primers 515F and 806R. The forward primer sequence was 5′-AATGATACGGCGACCACCGAGATCTACAC-TATGGTAATT-GT-GTGCCAGCMGCCGCGGTAA-3′, and the reverse primer was 5′-CAAGCAGAAGACGGCATACGAGAT-NNNNNNNNNNNN-AGTCAGTCAG-CC-GGACTACHVGGGTWTCTAAT-3′. These primers included the Illumina adapter sequences, a 12-bp Golay barcode (represented by 'N's) exclusive to the 3′ primer, pad, linker, and the locus-specific primer. PCR reactions were performed in duplicate and pooled. Amplicons were quantified using the Qubit dsDNA HS Assay Kit (Invitrogen) and pooled at equimolar concentrations. The final pooled library was purified using the GeneJET PCR Purification Kit (Thermo Scientific).