Marine microbial communities from Deepwater Horizon subsurface plume in Gulf of Mexico - 52-1 below plume
From Rivers et al. 2013: "RNA was extracted according to Poretsky et al. (2009). Six internal RNA standards (artificial mRNAs synthesized by in vitro transcription of DNA templates) were added as internal controls (1.4 _ 1010 transcripts per sample) (Satinsky et al., 2013). The two smallest standards (~200_nt) were not appreciably recovered on the solid phase extraction matrix used for RNA purification and were not considered further. RNA extracts were depleted of rRNA by creating sample-specific removal probes targeting the large and small subunits of archaeal, bacterial and eukaryotic rRNA genes using the DNA of each sample (Stewart et al., 2010). The protocol was modified by pooling probes from multiple DNA samples (see below) and including an additional round of probe removal. rRNA-depleted mRNA was amplified using the MessageAmp II bacterial kit (Life Technologies) and complementary DNA was synthesized using the universal Riboclone complementary DNA synthesis system (Promega, Madison, WI, USA). Complementary DNA was sheared ultrasonically to ~225_bp, and each sample was run on one lane of an Illumina Genome Analyzer IIx (Illumina Inc., San Diego, CA, USA) using 150-bp paired-end chemistry."