title: GSM8241690: Emergence, ventral skin, rep01; Lithobates sylvaticus; RNA-Seq
library_construction_protocol: We extracted and purified total RNA using a modified protocol for phase separation using TRI-reagent (Ambion, Invitrogen, Carlsbad, CA, USA) and the Direct-zol RNA Miniprep Plus Kit (Zymo Research, Irvine, CA, USA) for purification. Briefly, after thawing samples, we pipetted off the RNAlater preservative and immediately added 500 mL TRI-Reagent and a sterile steel bead. We briefly vortexed the sample, bead, and reagent before adding 500 mL more TRI-reagent and vortexing again for 10 min. Following vortexing we added 100 mL of cold 1-Bromo-3-chloropropane to each sample, shook each sample by hand for 20 s, and incubated for 15 min at room temperature. We then centrifuged samples at -2°C for 60 min at 16,000xg. After centrifuging, we checked samples to confirm that phase separation had occurred and transferred the aqueous (upper) RNA-containing phase to a sterile microcentrifuge tube for purification and DNase treatment, which we performed according to the manufacturer's suggested protocol. Total RNA samples were prepared for mRNA-sequencing using the Illumina Stranded mRNA Sample Preparation kit following the manufacturer's protocol (Illumina, San Diego, CA, USA). Libraries were validated for length and adapter dimer removal using the Agilent TapeStation 4200 D1000 High Sensitivity assay (Agilent Technologies, Santa Clara, CA, USA) then quantified and normalized using the dsDNA High Sensitivity Assay for Qubit 3.0 (Life Technologies, Carlsbad, CA, USA).