GSM4985016: Insular Cortex Input Rep 1; Mus musculus; RIP-Seq
Fourteen days after injection mice were sacrificed by cervical dislocation and the insular cortex was rapidly dissected in ice-cold Buffer B (1xHBSS, 4 mM NaHCO3, 2.5 mM HEPES [pH 7.4], 35 mM Glucose) with 100 mg/ml cycloheximide (Sigma). The dissected pieces were pooled in 3 groups of 5-6 brains each and transferred to a glass homogenizer (Kimble Kontes 20), and homogenized in 1.5 ml ice-cold Buffer C (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2) with 0.5 mM DTT (Sigma), 80 U/ml RNasin Plus (Promega), 40U/ml Superase-In (Life Technologies), 100 mg/ml cycloheximide, protease inhibitor cocktail (Roche) and 100 ng/ml GFP-Trap Protein (ChromoTek). Tissue samples were homogenized three times at 250 rpm and ten times at 750 rpm on a variable-speed homogenizer (Glas-Col) at 4°C. Homogenates were transferred to microcentrifuge tubes and clarified at 2,000xg for 10 min at 4°C. 140 μl each of 10% IGEPAL CA-630 (NP-40; Sigma) and 1,2-diheptanoyl-sn-glycero-3-phospho-choline (DHPC at 100 mg/0.69 ml; Avanti Polar Lipids) was added to the supernatant. The solutions were mixed and centrifuged again at 17,000xg for 15 min at 4°C. The resulting supernatants were transferred to new tubes and 50 μl of each cleared lysate was mixed with 50 μl Lysis Buffer (0.7 μl β-mercaptoethanol/100 μl Lysis Buffer; Agilent Absolutely RNA Nanoprep Kit) and stored at -80° for later preparation as input RNA. The remaining lysates (approximately 1.5 ml) were used for immunoprecipitation. The beads incubating with GFP antibodies were washed twice in Buffer A with 0.5 mM DTT, 80 U/ml RNasin Plus and 100 mg/ml cycloheximide before the cleared brain lysates were added. The immunoprecipitation was allowed to run at 4°C for 40 min. Beads were washed four times with Buffer D (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP40) with 0.5 mM DTT, 80 U/ml RNasin Plus and 100 mg/ml cycloheximide. Before removing the last wash solution the beads were moved to a new tube. After the final wash, RNA was eluted by adding 100 μl Lysis Buffer and purified using the Absolutely RNA Nanoprep Kit (Agilent). cDNA was amplified using SMARTer Ultralow Input RNA for Illumina Sequencing Kit and sequenced on an Illumina HiSeq2500 platform.