title: Sequencing of Fecal microbiota in Postpartum Cows with Different Calcium Metabolism
design: Fecal genomic DNA from days 1, 3, 7, 14, and 21 was extracted using the DNeasy PowerSoil Kit (cat. no. 47014; Qiagen, Hilden, Germany) for 16S rRNA and metagenome sequencing. DNA quantity and quality were assessed with a NanoDrop ND-2000 spectrophotometer (Thermo Scientific Inc., USA) and quality-checked via 1.0% agarose gel electrophoresis. The 16S rRNA gene V3V4 region was PCR-amplified in triplicate using primers 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR amplicons were cleaned with Vazyme VAHTS DNA clean beads (Vazyme Biotech Co., Ltd., Nanjing, China), pooled at equimolar ratios, and sequenced on an Illumina NovaSeq 6042 platform (Illumina, Inc., CA, USA).