title: GSM8858619: Pnoc_LepR_WT_Input_1; Mus musculus; RNA-Seq
library_construction_protocol: Immediately after sacrifice, hypothalamic samples from random fed mice (10–12-week-old) dissected out using a stainless-steel brain matrix (World precision instruments) and directly snap frozen in liquid nitrogen. Protein A Dynabeads (375 μl per IP, Invitrogen) were washed 3x in 0.15M KCl IP wash buffer (20 mM HEPES [pH 7.4], 5 mM MgCl2, 150 mM KCl, 1% NP40, 0.5 mM DTT and 100 μg/ml cycloheximide). Beads were subsequently resuspended in 275 μl 0.15M KCl IP wash buffer and loaded with 50 μg of 19C8 and 50 μg of 19F7 (Cat# Htz-GFP-19F7 and Htz-GFP-19C8, Memorial Sloan Kettering Monoclonal Antibody Facility) and rotated over night at 4°C. Pooled hypothalamic tissue (4 pooled animals per sample; 4 samples per group) was homogenized in lysis buffer (20 mM HEPES [pH 7.4], 5 mM MgCl2, 150 mM KCl, 0.5 mM DTT, 40 U/ml RNasin, 100 μg/ml cycloheximide, protease and phosphatase inhibitor cocktails [1 tablet of cOmplete mini EDTA-free protease inhibitor cocktail/7 mL and 2 tablets of PhosSTOP/10 ml]) 2x at 250 rpm, and 9x at 750 rpm on a rotating glass/teflon potter homogenizer (Potter S, Braun) at 4°C. Homogenates were transferred to microcentrifuge tubes (Nonstick, RNase-free microfuge tubes, 1.5 ml, Ambion, Invitrogen) and centrifuged at 2,000xg for 10 min at 4°C. Working on ice, the supernatant was transferred to a new tube, and 1/9 sample volume 10% NP-40 (final concentration: 1%) and 1/9 sample volume 300 mM DHPC (final concentration: 30 mM) was added. Solution was then incubated on ice for 2 min and before centrifuging at 17,000xg, 10 min at 4°C. The supernatant was moved to a new tube, and a 25 μl aliquot was removed, transferred to a new tube, flash frozen in liquid nitrogen and stored at 80°C for purification as input RNA. For immunoprecipitation (IP) 200 μl of antibody-bound magnetic beads were added to the remaining supernatant (800-1000 μl supernatant) and incubated at 4°C, 1h rotating. The magnetic beads were collected and resuspended in 1000 μl of 0.35 M KCl IP wash buffer (20 mM HEPES [pH 7.4], 5 mM MgCl2, 350 mM KCl, 1% NP40, 0.5 mM DTT, 100 μg/ml cycloheximide). Beads were washed 3x more in 1000 μl of 0.35 M KCl IP wash buffer, and then collected. The RNA was eluted by addition of buffer RLT (350 μL) to the beads, allowed to incubate at RT for 5 minutes and RNA was purified subsequently using the RNeasy Micro Kit (QIAGEN). RNA integrity was assessed using an Agilent 2100 bioanalyzer. Sequencing libraries were generated using the Illumina Nextera XT DNA sample preparation kit (San Diego, CA, USA), with 1 ng cDNA input.