16S rRNA gene sequencing of: cold seep metagenome: seep sediment
DNA was extracted using the Power Soil Pro DNA Isolation Kit. The V4-V5 region of the 16S rRNA gene was amplified using archaeal/bacterial primers with Illumina (San Diego, CA, USA) adapters on 5 end (515F 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-GTGYCAGCMGCCGCGGTAA-3 and 926R 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-CCGYCAATTYMTTTRAGTTT-3). Each product was barcoded with Illumina NexteraXT index 2 Primers that include unique 8-bp barcodes (P5 5-AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TCGTCGGCAGCGTC-3 and P7 5-CAAGCAGAAGACGGCATACGAGAT-XXXXXXXX-GTCTCGTGGGCTCGG-3). Products were run on 1.5% agarose gel and quantified by band intensity. Products were pooled and run on 1.5% low melt agarose gel and purified with Promegas Wizard SV Gel and PCR Clean-up System, Promega#A9281. Sample was sequenced by Laragen (Culver City, CA, USA) using MiSeq Reagent Kit v3 (600-cycle) #MS-102-3003 on Illuminas MiSeq platform with the addition of 15-20% PhiX. The length was MiSeq processed for 300bp or occasionally 250bp fragments.