Harvard Forest soil microbial gene expression

This project sequenced the metagenome and metatranscriptome of a temperate forest soil microbial community. Soil was collected from within a transition hardwood-white pine/hemlock forest in the Prospect Hill Tract of Harvard Forest (Massachusetts, USA; 42.54 N 72.18W; elevation: 385 m) on 27 September, 2010. Two cores were taken from 1-10 cm below the leaf horizon using a 10 mm diameter soil corer. These cores were homogenized, placed into 50 ml Falcon tubes, flash frozen in liquid nitrogen, and transported to the lab on dry ice. Visible pieces of plant material were removed from soil subsamples with sterile forceps. During plant removal, the subsamples were placed on a sterile surface, laid within a bed of dry ice. Total microbial DNA and RNA was then extracted from these subsamples using PowerSoil Total RNA and DNA isolation kits (MoBio), according to the manufacturer's protocol. Total DNA was quantified and used directly for pyrosequencing. Total RNA was further processed to reduce the proportion of ribosomal RNA transcripts via a custom subtractive hybridization procedure. Remaining total RNA was then linearly amplified, converted to cDNA, and used for pyrosequencing. Soil DNA and cDNA were sequenced with full plate runs on a Roche Genome Sequencer FLX instrument using Titanium chemistry.