title: Gut microbiome of control and antibiotics treated WT and iNOS knockout mice
design: V3-V4 region of 16S rRNA gene was amplified and gel extracted for metagenomic sequencing as described previously (Tandon et al., 2018). Metagenome libraries were prepared using V3-V4 region-specific primers. Approximately, 40 ng of DNA samples were amplified for 26 cycles of round 1 PCR using KAPA HiFi Hot-Start PCR Kit (KAPA Biosystems Inc., USA). The forward and reverse primers were used at the concentration of 5 M each. The amplicons were analyzed on 1.2% agarose gel. l l of diluted round 1 PCR  amplicons were used for Indexing PCR (Round 2). Here, the round 1 PCR amplicons were amplified for 10 cycles to add Illumina sequencing barcoded adaptors (Nextera XT v2 Index Kit, Illumina, USA). Round 2 PCR amplicons (sequencing libraries) were analyzed on 1.2% agarose gel. Illumina Adapter Sequences: 5-AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC and 5-CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG where [i5, i7] was unique dual index sequence to identify sample-specific sequencing data.