title: GSM7311320: 4 hpf polyA clutch A; Hydractinia symbiolongicarpus; RNA-Seq
library_construction_protocol: Frozen embryos in 1.5 ml eppendorf tubes were homogenized with a pestle in 500 µl of TRIzol (Invitrogen #15596026) (note that for later timepoints 24-72 h.p.f., some particulates remained after pestling). 100µl chloroform was added, vortexed for 15 sec, then centrifuged at 13,000 RPM at 4ºC for 15 minutes. The aqueous phase plus 340 µl of isopropanol and 1 µl of GlycoBlue (Invitrogen #AM9515) were added to a new tube and precipitated overnight at -20º C. To pellet, tubes were centrifuged at 13,000 RPM at 4º C for 30 minutes, supernatant removed, 500 µl of cold 75% ethanol added, and centrifuged at 13,000 RPM at 4º C for 15 minutes. Ethanol was drawn off with a P200, then P10 pipet and tubes left to air dry in a fume hood for 10 minutes. rRNA depletion was performed as per Phelps et al NAR 2021 using custom generated nuclear and mitochondrial rRNA antisense oligos. poly(A) selection was performed using NEB-Next Poly(A) mRNA Magnetic Isolation Module (NEB #E7490L). Strand-specific RNA-seq libraries were constructed using the NEB Ultra II RNA-seq library kit (NEB #E7765) according to manufacturer's protocol with the following parameters: 12-35 ng of rRNA-depleted or poly(A)+ selected RNA was fragmented at 94º C for 15 minutes in first-strand buffer, then subjected to half-reaction volume library prep with 5-fold dilution of adapters and 9 cycles of PCR amplification; Sera-Mag Select beads were used for cleanup steps (Cytiva 29343045).