454 run #3 (Eurofins MWG, cc4)
Globally distributed marine sediments (mostly from the top few cm) were slurried, amended with organic substrates and spirulina, pasteurized and incubated anoxically at 50°C. DNA from before and after 120 h of high temperature incubation was extracted from sediment slurries using the PowerSoil®DNA isolation kit (MO BIO Laboratories, Inc.) according to the manufacturer’s instructions. Bacterial 16S rRNA gene amplicons were obtained and supplied with barcodes and pyrosequencing adaptor sequences using a two-step PCR approach with low cycle numbers (20+5) and pooling of triplicate PCR reactions to reduce variability associated with barcoded pyrosequencing primers [3]. The PCR primers targeting most bacteria (909F: 5’-CCACTCAAAKGAATWGACGG-3’, 1492R: 5’-NTACCTTGTTACGACT-3’) and conditions were described previously (Berry et al., Appl Environ Microbiol, 2011. 77(21): p. 7846-9.). Final PCR products were purified using the Agencourt® Ampure® XP system (Beckman Coulter) and the DNA concentration was determined using a Quant-iTTM PicoGreen® dsDNA Assay (Invitrogen). Amplicons were then pooled and sequencing was performed on a GS FLX instrument using Titanium chemistry (Roche) by Eurofins MWG Operon (Ebersberg, Germany).