near rep 2_estuarine, october
Water samples were filtered through a 5 m, 47-mm Nuclepore filter, and immediately flash frozen in liquid nitrogen. To extract RNA, cells were homogenized in a custom guanadidium thiocyanate buffer in a bead beater using MoBio lysing matrix E and vortexed for 15 s, then RNA was extracted from the lysate using a TRIzol procedure followed by a Qiagen RNeasy column clean up. RNA was checked for quality using a Fisher nanodrop (minimum quantity = 25 ng RNA per l), and quantity using an Agilent bioanalyzer with a minimum RIN of 7 (lowered to accommodate the multiple taxa within the extraction and the low yield).  Samples were prepared for sequencing at the Ramaciotti Centre for gene function analysis using Illuminas TruSeq stranded mRNA kit with extended PCR to compensate for the low RNA yield.  Samples were run on Illuminas NextSeq 500 1X75bp using a high output protocol.