GSM8415358: VP2.6_TRAP; Mus musculus; RNA-Seq
The ventral pallidum from 7-10 adult mice was dissected into ice cold buffer (20 mM HEPES (pH 7.3), 150 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 100 μ g/mL cycloheximide, EDTA-free protease inhibitor cocktail, 5 μL/mL RNasin and 2 μL/mL Superasin). Samples were homogenized and centrifuged for 10 min at 20,000g, 4°C. NP-40 and 1, 2-diheptanoyl-sn-glycero-3-phosphocholine were added to the supernatant at final concentrations of 1 % and 30 mM, respectively. 200 µL of the sample was removed and 500 µL of Trizol was added to constitute the Input fraction. Streptavidin Dynabeads (300 µL, Invitrogen Cat#65601) pre-conjugated with Protein L (ThermoFischer Cat# 29997) and Anti-HA antibody (Biolegend Cat# 901513) in homogenization buffer were added to the remaining sample. Beads were collected on a magnet at 4°C and washed for 5 cycles with 1ml 350mM KCl buffer before resuspension in 700 µL of Trizol for the TRAP fraction. RNA was isolated following the Zymo RNA Clean & Concentrator 5 protocol with on-column DNase digestion. Samples were prepared according to library kit manufacturer's protocol, indexed, pooled, and sequenced on an Illumina NovoSeq