Rnase h buffer

To distinguish the size difference between the two Adh isoforms, the total RNA of each sample was treated with RNaseH prior to RNA blot analysis. A total of 15 µg Trizol-extracted RNA was added to 1x RNaseH buffer (New England Biolabs, Ipswich, MA). Next, a site-specific DNA oligo (See Table 1 for sequence) was annealed to RNA by heating to 52° and slowly cooling to 25°. The RNA-DNA hybrid strands were incubated with 1 U RNaseH (New England Biolabs) for 1 hr at 37°. RNA was extracted in phenol:chloroform (1:1) and precipitated in isopropanol with 0.3 M sodium acetate overnight at -20°.

To enrich for mRNA reads within a DNA library that was constructed using random priming, we developed an in-house approach to deplete ribosomal reads. Probes hybridizing to ribosomal RNA sequences of the bacterial species used in this study were designed (using previously designed software^{19 (link)}). Multiple reactions (depending on the yield of the in vitro transcription reaction) each containing 500 ng of RNA, probes, and hybridization buffer were prepared as follows (using protocols adapted from ref. ^{19 (link)}): 500 ng of in vitro transcribed RNA, 3 µg of rRNA probes, 0.6 μl 5 M NaCl, 1.5 μl 1 M Tris-HCl and nuclease-free water up to 15 μl. Hybridization was then performed using the following temperature programme: 95 °C for 2 min and 0.1 °C s⁻¹ ramp down to 25 °C, 25 °C for 5 min. Following rRNA probe hybridization, 6 μl RNase H mix consisting of 3 μl of 10× RNase H buffer (NEB B0297), 2 μl of thermostable RNase H (NEB M0523S) and 1 μl of RNase H were added to each tube. The reactions were incubated for 45 min at 50 °C to digest the rRNA–DNA hybrids. Following rRNA digestion, the DNA probes were degraded by adding 3 μl of 10× DNase I buffer, 3 μl of DNase I and incubating for 45 min at 37 °C. The rRNA-depleted RNA library was purified with a 2× SPRI reaction and eluted in 25 μl of nuclease-free water.

Total RNA from ovaries and testes was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. The quality and amount of purified RNAs were measured using a NanoPhotometer P330 (Implen, Germany).
From the purified fraction, 2S ribosomal RNA (rRNA) was depleted by annealing 5 μg of the elute to complementary oligonucleotide sequences (10 pmol/μl), 5′- AGTC​TTA​CAA​CCC​TCA​ACC​ATA​TGT​AGT​CCA​AGC​AGC​ACT -3′, in RNase-H buffer (New England Biolabs). The DNA/RNA hybrids were removed by treatment with RNase-H (New England Biolabs) for 30 min at 37°C.
Samples depleted of 2S rRNA were loaded onto 8 M urea–polyacrylamide gel (12%) and separated in 0.5 TBE buffer. Gel areas within the range of 20–30 nt (DynaMarker®, DM253, BioDynamcs Laboratory Inc.) were excised, and RNAs were eluted in 0.3 M sodium acetate overnight and precipitated in the presence of 80% (v/v) ethanol and 0.5–1 μg/μl glycogen (Nacalai). Pellets were rinsed twice in 80% (v/v) ethanol and dissolved in RNase-free water.