BA4A-270m: 2H Incubation
PCR of 16s was performed in duplicate using 515F/806R primers (V4 amplification), as described by the Earth Microbiome Project and using Platinum II Hot Start Master Mix (ThermoFisher) on SimpliAmp thermocyclers (ThermoFisher).  After confirming amplification via gel electrophoresis, amplicons were cleaned and normalized using the SequalPrep Normalization Kit from ThermoFisher. Pooled libraries were quantified using both the Qubit ds DNA High Sensitivity Assay (ThermoFisher) and the KAPA Library Quantification Kit for Illumina platfrom (Roche). The library was then sequenced on an Illumina MiSeq using a v2 500-cycle PE reagent kit at the Center for Microbial Exploration at the University of Colorado-Boulder.