title: Healthy broiler chicken jejunum microbiome 2
design: Total DNA was extracted from jejunal contents (200 mg/sample) using a QIAamp Fast DNA Stool Mini kit (Qiagen Inc.). The DNA concentration was measured by a NanoDrop spectrometer (Thermo Scientific, USA). The 16S amplicon PCR (targeting the V3-4 region) was performed as per the Illumina 16S metagenomic sequencing library preparation protocol. The PCR products were purified using AMPure XP magnetic beads (Beckman Coulter, Inc, CA, USA). The size of the amplicons was confirmed by gel electrophoresis. Sequence libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, USA) with 24 or 96 indexes as per the manufacturers instructions. The libraries were cleaned up with AMPure XP magnetic beads and analyzed by a bioanalyzer using a high sensitivity DNA kit (Agilent, Santa Clara, CA). The concentration of the amplicon sequence libraries was measured using the Qubit dsDNA BR (broad range) assay kit (Thermo Fisher Scientific, Waltham, USA). The normalized libraries were pooled, denatured with 0.2N NaOH, diluted using H1 hybridization buffer and loaded onto the MiSeq V3 (600 cycles) cartridge. Sequencing was performed using the Illumina MiSeq platform (Illumina, San Diego, US) as per the companys protocol. The sequence quality was examined in real time using the Sequencing Analysis Viewer (SAV) V1.8.