GSM9097124: mod(mdg4)[c13]_male_rep2; Drosophila melanogaster; OTHER
Total RNA was extracted from imago males of the control y[1]w[1118] line and the y[1]w[1118]; mod(mdg4)[c13] line. An aliquot of 50 µL of hydrophilic streptavidin magnetic beads (NEB) were washed with 500 µL of wash/binding buffer (0.5 M NaCl, 20 mM Tris-HCl pH 7.5, 1 mM EDTA), magnetically pelleted, and subsequently blocked with 100 µL of wash/binding buffer containing 250 ng/µL of sonicated salmon sperm DNA. After an additional wash with 500 µL of wash/binding buffer, the beads were incubated for 15 min at room temperature, with rotation, in 25 µL of wash/binding buffer containing 200 pmol of a biotinylated oligonucleotide complementary to exon 4 (8 pmol/µL, bio-ACATTTGCGGAGGTGCTTGTCTTGGA) RNA. This incubation was followed by two subsequent washes with 100 µL of wash/binding buffer. Total RNA (100 µg) was denatured at 65°C, chilled on ice, and then added to the beads. This mixture was incubated at 55°C for 30 min with agitation. Five sequential washes followed. The first two washes used wash buffer (1 M NaCl, 20 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.05% Tween-20) at 55°C. The third wash employed the same buffer but without Tween-20. The fourth wash used low salt buffer (150 mM NaCl, 20 mM Tris-HCl (pH 7.5), and 1 mM EDTA), and the fifth was with ice-cold low salt buffer. RNA was eluted by adding 200 µL of TRI Reagent® (MRC) and then extracted following the manufacturer's protocol with glycogen added prior to isopropanol precipitation. Preparation of RNA-seq libraries followed the manufacturer's instructions for the NEBNext® Ultra RNA Library Prep kit (NEB, USA).