16S microbial tag sequencing of methane seep sediment_9108
DNA was extracted using the Power Soil DNA Isolation Kit 12888 accordingto manufacturer (Qiagen, Germantown, MD, USA) directions. The V4-V5 region ofthe 16S rRNA gene was amplified using archaeal/bacterial primers with Illumina (SanDiego, CA, USA) adapters on 5 end (515F 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-GTGYCAGCMGCCGCGGTAA-3 and926R 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-CCGYCAATTYMTTTRAGTTT-3). Each product was barcoded with Illumina NexteraXT index 2 Primers that includeunique 8-bp barcodes (P5 5-AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TCGTCGGCAGCGTC-3 and P7 5-CAAGCAGAAGACGGCATACGAGAT-XXXXXXXX-GTCTCGTGGGCTCGG-3). Products were purified using Millipore-Sigma (St. Louis,MO, USA) MultiScreen Plate MSNU03010 with vacuum manifold and quantified usingThermoFisherScientific (Waltham, MA, USA) QuantIT PicoGreen dsDNA Assay KitP11496 on the BioRad CFX96 Touch Real-Time PCR Detection System. Barcodedsamples were combined in equimolar amounts into single tube and purified withQiagen PCR Purification Kit 28104 before submission to Laragen (Culver City, CA,USA) for 2 x 250bp paired end analysis on Illuminas MiSeq platform with PhiXaddition of 15-20%