GSM6268565: Stage 9 CHX pou5f3.3, sox3 MO rep C1; Xenopus laevis; RNA-Seq
Two embryos per sample were snap frozen and homogenized in 500ul of TRIzol Reagent (Invitrogen #15596026) followed by 100ul of chloroform. Tubes were spun at 18,000 x g at 4C for 15 minutes, the aqueous phase was transferred to a fresh tube with 340 ul of isopropanol and 1 ul of GlycoBlue (Invitrogen #AM9515), then precipitated at -20C overnight. Precipitated RNA was washed with cold 75% ethanol and resuspended in 50ul of nuclease-free water. Concentration was determined by NanoDrop. rRNA depletion was performed as per Phelps et al NAR 2021: 1ul of antisense nuclear rRNA oligos and 1ul of antisense mitochondrial rRNA oligos (final concentration 0.1 uM per oligo) were combined with 1ug of total RNA in a 10ul buffered reaction volume (100mM Tris-HCl  pH 7.4, 200mM NaCl, 10mM DTT), heated at 95C for 2 minutes and cooled to 22C at a rate of 0.1C/s in a thermocycler. Next, 10U of thermostable RNaseH (NEB #M0523S) and 2uL of provided 10X RNaseH buffer were added and volume brought to 20uL with nuclease-free water. The reaction was incubated at 65C for 5 or 30 minutes, then 5U of TURBO DNase (Invitrogen #AM2238) and 5uL of provided 10x buffer was added, volume brought to 50uL with nuclease-free water and incubated at 37C for 30 minutes. The reaction was purified and size selected to >200 nts using Zymo RNA Clean and Concentrator-5 (Zymo #R1013) according to manufacturer's protocol, eluting in 10uL of nuclease-free water. The WT Stage 5 sample was also depleted of mitochondrial COX2 and COX3 mRNA as part of Phelps et al NAR 2021. Strand-specific RNA-seq libraries were constructed using NEB Ultra II RNA-seq library kit (NEB #E7765) according to manufacturer's protocol with fragmentation in first-strand buffer at 94C for 15 minutes. Following first and second strand synthesis, DNA was purified with 1.8X AmpureXP beads (Beckman #A63880), end repaired, then ligated to sequencing adaptors diluted 1:5. Ligated DNA was purified with 0.9X AmpureXP beads and PCR amplified for 8 cycles, then purified again with 0.9X AmpureXP beads. Libraries were verified by Qubit dsDNA high sensitivity (Invitrogen #Q32851) and Fragment Analyzer prior to multiplexed sequencing at the Health Sciences Sequencing Core at Children's Hospital of Pittsburgh.