GSM8730232: Il-17rb_replicate_2; Mus musculus; RNA-Seq
The S1DZ tissue was homogenized with a glass/Teflon power-driven Potter-Elvehjem homogenizer in an ice-cold lysis buffer (150 mM KCl, 10 mM HEPES [pH 7.4], 5 mM MgCl2, 0.5mM dithiothreitol, 100 μg/mL cycloheximide, 20 U/µL SUPERaseIn RNase Inhibitor, 40 U/µL RNasin Plus Rnase Inhibitor, and EDTA-free protease inhibitors).  Following homogenization, samples were centrifuged at 2,000 x g at 4°C for 10 min and the supernatant was removed to a new tube.  NP-40 (final concentration 1%) and 1,2-Diheptanoyl-sn-glycero-3-phosphocholine (DHPC, final concentration 15 mM) were subsequently added and samples were incubated on ice for 5 min.  Samples were centrifuged at 13,000 x g at 4°C for 10 min and the supernatant was removed to a new tube.  Streptavidin Dynabeads (ThermoFisher Scientific, Waltham MA) coated with biotin-linked mouse anti-GFP antibodies were then added to this supernatant and the samples were incubated overnight at 4°C with end-over-end rotation.  Beads were collected on a magnetic rack and washed three times with wash buffer (350 mM KCl, 10 mM HEPES [pH 7.4], 5 mM MgCl2, 0.5 mM dithiothreitol, 100 μg/mL cycloheximide, 1% NP-40).  RNA was subsequently purified using the Absolutely RNA Isolation Nanoprep kit (Agilent Technologies, Santa Clara CA). Libraries were constructed using the Takara Bio SMART-Seq v4 kit (Takara Bio USA, Inc.).