GSM3230580: E36 high TBPH exposure; Fundulus heteroclitus; RNA-Seq
Total RNA was isolated from embryos using the MagMAX™-96 Total RNA Isolation kit (ThermoFisher, Waltham, MA) following the manufacturer's protocol supplied with the kit, except as noted below. Homogenization of embryos was performed in 1.5 ml microcentrifuge tubes using a Bullet Blender© Storm 24 homogenizer (Next Advance, Troy, NY) with a 3.2 mm stainless steel bead added to each sample. Each embryo was homogenized in 100 µl of MagMax Lysis/Binding solution supplemented with 0.7 µl β-mercaptoethanol. Contaminating DNA was removed from samples using the supplied Turbo DNase. Both the duration of DNase treatment and incubation temperature were increased, to 20 min and 37 °C, to increase the effectiveness of digestion. RNA was eluted from the magnetic beads using 25 µl of Elution Buffer. During the elution step, samples were incubated at 37 °C Libraries were prepared (n = 16 per treatment) using the TruSeq Stranded mRNA Library Prep Kit for Neoprep (Illumina, San Diego, CA) according to the manufacturer's protocol.  One hundred ng RNA was used as input into the automated NeoPrep System (Illumina, San Diego, CA).  The NeoPrep is able to prepare libraries for up to 16 samples.  Samples were blocked by treatment for a multiplex level of 16 samples.  This level of multiplexing provides a theoretical sequencing depth of 18.75 M sequences per sample. Following library preparation, individual samples were quantified using the Qubit fluorometer (Thermofisher), normalized to 10 nM and pooled
GEO Accession
GSM3230580