GSM4813825: Stage 5 rRNA, COX2, COX3 deplete, 65ºC; Xenopus laevis; RNA-Seq
For X. laevis, 2 embryos were pooled for RNA extraction; for D. rerio, 20 embryos or 10 fin clips were pooled. Samples were snap frozen in a 1.5ul tube and homogenized with a pestle 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 -80C for 1 hour. Precipitated RNA was washed with cold 75% ethanol and resuspended in 50ul of nuclease-free water. Concentration was determined by NanoDrop. RNA was stored at -80C until use. For rRNA depletion, 1ul of antisense nuclear rRNA oligos (final concentration 0.4 uM per oligo) and 1ul of antisense mitochondrial rRNA (plus any other targeted gene) 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 minutes (standard) or 45C for 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 Clean and Concentrator-5 (Zymo #D4013) according to manufacturer's protocol, eluting in 10uL of nuclease-free water. For poly(A) selection, the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490L) was used according to the manufacturer's protocol: 1 ug of total RNA was denatured at 65C for 5 minutes then hybridized to buffered dT magnetic beads at room temperature for 2 minutes. Selected RNA was eluted in 50uL of Tris buffer at 80C for 2 minutes and rehybridized to the same beads for a second round of selection at room temperature for 2 minutes. Re-selected RNA was eluted in a final volume of 17 uL of Tris buffer. 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 (paired end 38/37bp) on an Illumina NextSeq 500 at the Health Sciences Sequencing Core at Children's Hospital of Pittsburgh.
GEO
GSM4813825