Chicken Cecal Microbiome Analysis via 16S rRNA Sequencing

All the samples collected during the chicken experiments (n = 58) were selected for microbiota analysis. Genomic DNA was extracted from 150 to 200 mg of cecal contents using the PureLink Microbiome DNA Purification Kit (Life Technologies, Invitrogen Corp.) and combined with RNAse treatment (10 units/h). After quality control with nanodrop, the 16S rRNA V4-V5 variable region was amplified, purified, and sequenced. Amplicon libraries were prepared by using Phusion® High-Fidelity PCR Kit (New England Biolabs Inc., Ipswich, MA, United States), as previously described (Kumar, 2015 ; Deblais et al., 2018 (link), 2019 (link); Kumar et al., 2018 (link); Srivastava et al., 2020 (link)). PCR products were cleaned using AMPure XP PCR (Beckman Coulter Inc., Beverly MA, USA) and were sequenced using Illumina MiSeq 300-base paired-end kit at the Molecular and Cellular Imaging Center.¹ Sequencing raw data files are publicly available at NCBI Bioproject #PRJNA1023035.