title: GSM2187968: C4_4; Schmidtea mediterranea; RNA-Seq
library_construction_protocol: Live embryos were dissected out of egg capsules in 1x Holfreter’s buffer (3.5 g/L NaCl; 0.2 g/L NaHCO3; 0.05 g/L KCl; 0.2 g/L MgSO4; 0.1 g/L CaCl2; 1.0 g/L dextrose, pH 7.0-7.5) for Stage (S) S2-S7 egg capsules, or 1x Montjuic water for S8 hatchlings.  Yolk samples were obtained from 8 d egg capsules that contained neither spherical nor elongating embryos.  Single embryos were  transferred into microfuge tubes containing 200 µl TRIzol reagent (Thermo Fisher, item #15596-018), homogenized by pipetting, and stored at -80˚C. Single animal samples of intact C4 adults and virgin, sexually mature adults were homogenized in 1.0 mL TRIzol using an IKA Ultra Turrax T 25 Basic tissue disruptor prior to storage at -80˚C.  Total RNA extraction was performed in 1.0 mL TRIzol per sample according to the manufacturer’s protocol, following the recommendations for working with small amounts of tissue. PolyA-selected, single stranded RNA-Sequencing libraries were prepared for four biological replicates per stage using the Illumina TruSeq RNA Sample V2 kit (item # RS-122-2001 and RS-122-2002), starting with 500 ng total RNA per sample (C4, virgin sexual adult (SX), Y, S4, S5, S6, S7, S8), or 100 ng total RNA per sample (S2, S3).