GSM4073693: FM rep1 (FH rep1); Drosophila melanogaster; RNA-Seq
Samples were fixed in 1 ml of PBS containing 1% formaldehyde (Sigma-Aldrich) and 0.5% Igepal CA-630 (Sigma-Aldrich) for 1 h on ice, then, incubated for 5 min after addition of 140 μl of 2 M glycine. Samples were then rinsed three times with ice cold PBS and homogenized by sonication in 2 ml of homogenization buffer (150 mM NaCl, 50 mM HEPES buffer at pH 7.6, 1 mM EGTA, 15 mM EDTA and 10% glycerol) supplied with 8 mM vanadyl ribonucleoside complex (Sigma-Aldrich), 50 U/ml SUPERase-In (Thermo Fisher Scientific), and 1 tablet/50 ml protease inhibitor cocktail tablet (Roche). After centrifugation at 13,000 g for 30 min, the supernantant was incubated with anti-FLAG-M2 affinity agarose beads (Thermo Fisher Scientific) at 4°C O/N. The beads were then washed four times with homogenization buffer. And the RNAs bound to the pAbp were recovered in 100 ml of elution buffer (50 mM Tris–HCl, pH 7.0, 10 mM EDTA, 1.3% SDS and 50 U/ml SUPERase-In) by proteinase K (40 μg/mL) treatment 2h at 60°C and O/N at 65°C after addition of NaCl (200mM). Eluant was then mixed with 4V of Trizol (Thermo Fisher Scientific) and 1V of chloroform (Sigma-Aldrich) with vigorously shaking, incubated at room temperature for 10 min, and then centrifuged at 12,000 g for 15 min at 4°C. The supernatant was transferred and mix with 2.5V of isopropanol, chilled at -80°C O/N, and centrifuged at 12,000 g for 30 min at 4°C. The RNA pellet was rinsed twice with 70% ethanol and then dissolved in RNase free water. RNA libraries were generated with standard Illumina protocols