GSM4649353: Day sample - Replicate 3; Stylophora pistillata; RNA-Seq
Total RNA was extracted with Trizol Reagent (Invitrogen). Frozen coral samples were ground into powder in a cryogrinder (Freezer/Mill 6770, Spex Sample Prep) and dissolved in 20 mL Trizol Reagent. Samples were vortexed and incubated at room temperature (RT) for 5 min. Three centrifugations (10 min, 15,000 × g, 4°C) were performed to remove the skeleton. We added 4 mL chloroform to the last supernatant. Centrifugation (15 min, 12,000 × g, 4°C) was performed to separate the two phases (aqueous and phenol-chloroform). RNA remained exclusively in the aqueous phase. The aqueous phase was subjected to another chloroform extraction (v/v). We added 10 mL isopropanol to the second aqueous phase, which was vigorously shaken and incubated for 10 min at RT. Centrifugation (15 min, 12,000 × g, 4°C) was performed to pellet the RNA. The pellet was resuspended in 5 mL 70% ethanol, vortexed vigorously, centrifuged for 5 min at 7,500 × g and 4°C, dried for about 10 min at RT, and then resuspended in 100–200 μL RNase-free water The concentration and quality of total RNA were assessed with a Bioanalyser and RNA 6000 nano kit. mRNA was prepared with the MicroPoly(A) purist kit (AM1919; Applied Biosystems)
GEO Accession
GSM4649353