seafloor
DNase-treated RNA (1 g reactions) was amplified by in vitro transcription using the MessageAmp II aRNA Amplification Kit (Ambion). The aRNA generated was poly-A tailed and precipitated according to the kit instructions using NH4OAc and ethanol. RNA (0.25 g) was resuspended in 5 L DEPC-treated water and reversed transcribed to double-stranded cDNA using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). cDNA was quantified with a Qubit fluorometer using a dsDNA BR Assay kit (Q32850, ThermoFisher), and quality was assessed using High Sensitivity D1000 ScreenTape (5067-5584, Agilent) and reagents (5067-5585, Agilent) on an Agilent 2200 TapeStation. Sequencing libraries were prepared using an Ovation Ultralow DR Multiplex System (0331-32, NuGEN) with some modifications to the manufacturers protocol. Briefly, cDNA was sheared in a total volume of 130 l in a microTUBE AFA Fiber Snap-Cap (520045, Covaris) using a Covaris M220 focused-ultrasonicator with the following settings: Peak Incident Power = 50; Duty Factor = 2.0; Cycles per burst = 200; Treatment Time = 160 s. Sheared DNA was cleaned up using a MinElute Reaction Cleanup kit (28204, QIAGEN). End Repair, ligation and amplification steps were performed according to manufacturers protocol, but ligation and amplified library purification steps were performed with a QIAquick PCR Purification kit (28104, QIAGEN). Amplified libraries were size-selected on a BluePippin (Sage Sciences) using a 1.5% agarose gel cassette (BDF1510, Sage Sciences) with a size range of 300  500 bp. The size-selected libraries were cleaned up using a MinElute Reaction Cleanup kit, assessed for quality on an Agilent 2200 TapeStation using D1000 ScreenTape and reagents, and quantified with a KAPA Library Quantification Kit (KK4824, KAPA). Libraries were multiplexed and sequenced on an Illumina NextSeq 500 sequencer using a High Output v1 NextSeq kit (FC-404-1004, Illumina).