title: Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill distal plume MT
design: Samples intended for RNA extractions were placed in RNAlater to prevent RNA degradation. As described in Mason et al. (2012) total RNA was extracted from the proximal and distal plume stations, as well as from the uncontaminated sample from plume depth, as using the protocol in (DeAngelis et al, 2010). The quantity and quality of extracted RNA was checked using a Bioanalyzer .  The RNA integrity number (RIN) was ~9 on a scale of 1-10, with 10 indicating that no degradation had occurred. Insufficient RNA was obtained from the uncontaminated sample for downstream processing. Total RNA from the proximal and distal plume stations was amplified using the Message Amp II-Bacteria Kit and first strand synthesis of cDNA from the resulting antisense RNA was carried out with the SuperScript III First Strand Synthesis System. The SuperScript Double-Stranded cDNA Synthesis Kit was used to synthesize double stranded cDNA.  cDNA was purified using a QIAquick PCR purification kit. Poly(A) tails were removed by digesting purified DNA with BpmI for 3 h at 37C. Digested cDNA was purified with QIAquick PCR purification kit. cDNA were amplified by emulsion PCR. Amplified cDNA was sequenced using the Illumina GAIIx. sequencing platform.