title: Experimental cryo hole P111 with treatment AL after 2 seasons
design: Frozen ice layer around each collected sediment was rinsed with filtered, deionized water before being melted in a presterilized (70% ethanol and UV light at 4C for 12-24 h) beaker. When melted, the sediments were homogenized and a subset ~0.3 g was used for DNA extraction with the DNeasy Power Soil DNA Extraction Kit (Qiagen, Hilden, Germany) according to manufacturers directions. The extracted DNA was measured for concentration using a Qubit fluorometer (Invitrogen Corp., CA, USA) and stored at -20C till amplifying and sequencing. To describe bacterial communities, we applied 16S metabarcoding using standard Earth Microbiome Project primers (515f-806r) (39, 40) and PCR conditions. Amplified DNA was pooled and normalized to equimolar concentrations using SequalPrep Normalization Plate Kit (Invitrogen Corp., CA, USA). Libraries were sequenced on an Ilumina MiSeq at 2250 bp chemistry for the 16S library by the Biofrontiers Sequencing Facility at the University of Colorado Boulder.