16S rRNA Amplicon Sequencing of Gut Microbiome

16S rRNA V3-V4 hyper variable region sequencing (Clevergene, Bangalore, India) was performed on the faecal samples collected from the mice prior to the sacrifice. The sequencing employed KAPA HiFi HotStart Ready Mix kit and 341F and 785R as the primers. Sequencing libraries were created by running, an additional 8 cycles of DNA polymerase chain reaction (PCR) using Illumina bar-coded adapters. The sequencing data was generated by Illumina MiSeq software (USA). GREENGENES v.13.8-99 database, was employed to filter contigs and their organization into operational taxonomic units (OTU). The abundance of these OTU was estimated Fisher's exact test was used to determine the statistical significant differences in OTU abundance between samples (Schierová et al. 2020 (link); Peng et al. 2020 (link)).