RNA-seq of Ectocarpus sp7
Ectocarpus specimens were grown at 14C in Provasoli-enriched NSW a 12h/12h d/night cycle and 20 mol photons m2s1 irradiance, as described previously (Coelho et al. 2012). Snap-frozen algae were dry-grinded with a pestle in liquid N2/dry ice and mixed with 750 mL of freshly prepared RNA-extraction buffer (100 mM Tris-HCl pH8,0 (Thermo Fisher AM9856); 1,4 M NaCl; 2% CTAB (Sigma Aldrich, 52365-50G); 20 mM EDTA pH8,0; 1% b-mercaptoethanol; 2% Polyvinylpyrrolidon (Thermo Fisher AM9690) preheated to 65C. Then 250 mL 5M NaCl were added into the tubes. An equal volume of chloroform: isoamylalcohol (24:1) was added and mixed well followed by centrifugation at 10000g for 15 minutes at 4C. The aqueous phase was removed into RNAse-free tubes and extracted again with 250 mL pure ethanol and an equal volume of chloroform: isoamylalcohol (24:1) as before. RNA was precipitated by adding LiCl (Thermo Fisher, AM9480) to a final concentration of 4M, together with 1% volume of b-Mercaptoethanol, mixing and incubating at -20C overnight. The RNA was pelleted by centrifugation at full speed (>18000g) for 45 minutes to 1 hour at 4C. RNA was washed with 70% cold Ethanol and the pellet was air dried for 3-5 minutes and then the RNA was dissolved in 30 mL RNAse free H2O. Residual DNA was eliminated using the TURBO DNase Kit (Thermo Fisher, AM1907) according to the manufacturers instructions. The final RNA concentration and size distribution were determined using a Qubit (RNA BR Assay Kit, Invitrogen, Q10210) and an RNA Nano bioanalyzer (Aglient, 5067-1511). The RNA-seq libraries were prepared using commercially available kits according to the manufacturers instructions. Poly-A selection for mRNA enrichment was performed using the corresponding NEB kit (#E7490) followed by library preparation using the low input RNA library prep kit from NEB (#E6420) since large amounts of material were not possible to obtain. The Qubit 1x dsDNA HS assay kit (Invitrogen, Q33230) was used to determine the final DNA concentration of the libraries and the DNA high-sensitive Kit (Aglient, 5067-4626) was employed for bioanalyzer analysis to evaluate the distribution of insert sizes.