Rnaseh and e coli dna polymerase

Methods for microbial community DNA and mRNA sequencing were as previously described ^{44 (link),45 (link)}. Fecal pellets collected from mice before or after LPS treatment (pooled from 2 and 3 days post-injection) were kept at −80°C until DNA and RNA isolation using the guanidinium thiocyanate/cesium chloride gradient method ^{46 (link)} as described, except that crude particles were removed by centrifugation prior to overlaying the gradient. RNA was subjected to DNase treatment (Ambion), purification using MEGAClear columns (Ambion), and rRNA depletion via subtractive hybridization (MICROBExpress, Ambion, in addition to custom depletion oligos). The presence of genomic DNA contamination was assessed by PCR with universal 16S rDNA primers. cDNA was synthesized using SuperScript II and random hexamers (Invitrogen, Carlsbad, CA), followed by second strand synthesis with RNaseH and E.coli DNA polymerase (New England Biolabs). Samples were prepared for sequencing with an Illumina HiSeq instrument after enzymatic fragmentation (NEBE6040L/M0348S). Libraries were quantified by quantitative PCR (qPCR) according to the Illumina protocol. qPCR assays were run using ABsoluteTM QPCR SYBR Green ROX Mix (Thermo Scientific) on an Mx3000P QPCR System instrument (Stratagene, La Jolla, CA). The size distribution of each library was quantified on an Agilent HS-DNA chip (Agilent, Santa Clara, CA).