16S sequence of Human Milk Meta Genome: Subject B004 using own pump
1.25 mL milk was thawed for 5 min at 37C, then centrifuged at 13,000  g for 10 min at 4C. The lipid layer and supernatant were discarded, and then the remaining cell pellet was resuspended in 500 L TE50 buffer, followed by enzymatic lysis for 1 h at 37C. Total DNA was extracted from the resuspended pellet following centrifugation using QIAamp DNA Mini Kits (Qiagen) following the manufacturer's instructions, with minor modifications. Both positives and negatives were included as controls. The V1V3 hypervariable region of the bacterial 16S rRNA gene was amplified using a dual-barcoded, 2-step, 30-cycle PCR protocol. For the first step, a 7-fold degenerate forward primer targeting position 27 and a reverse primer targeting position 534 (positions numbered according to the Escherichia coli rRNA) were used. Amplicon quality was assessed using the QIAxcel DNA screening cartridge (Qiagen), with quantity assessed using a Qubit 2.0 fluorometer and the DS High Sensitivity Assay (Thermo Fisher Scientific). Amplicons (50 ng) from each sample were pooled and sent to the Institute for Bioinformatics and Evolutionary Studies Genomics Resources Core at the University of Idaho for additional processing and sequencing.