title: Molecular profiling of hypothalamic leucine-sensing neurons via PhosphoTRAP
design: Molecular profiling of hypothalamic leucine-sensing neurons via PhosphoTRAP
library_construction_protocol: To identify enriched transcripts in mediobasal hypothalamic neurons activated or inhibited by the administration leucine compared to aCSF, PhosphoTRAP assay was performed. 45 min after the MBH injection, mice were culled by overdose of anaesthetic (300 mg/kg I.P. sodium pentobarbitone; Euthatal Solution for Injection, Dopharma Research B.V.). The MBH was micro-dissected in an ice-cold Buffer B (Hanks Balanced Salt Solution (HBSS) with 2.5 mM of HEPES at pH 7.4, 4 mM of NaHCO3, 35 mM of glucose, and 100 mg/mL of cycloheximide in methanol) under a 10x dissecting microscope. The hypothalami of 5 mice were pooled as a single sample resulting in 4 replicates per experimental condition (i.e. aCSF vs. Leu). Pooled hypothalami were manually homogenized in a 1ml of buffer C (10 mM of HEPES at pH 7.4, 150 mM of KCl, 5 mM of MgCl2, 100 nM of calyculin A, 2 mM of DTT, 100 U/mL of RNasin, 100 mg/mL of cycloheximide, and Roche protease and phosphatase inhibitor cocktails) and clarified by centrifugation at 2,000 g at 4°C for 10 min. The supernatants were transferred to a new tube. 70μl of 10% NP40 and 70μl of DHPC (1,2-diheptanoyl-sn-glycero-3-phosphocholine) were added, the samples were then mixed by inversion and allowed for 2 min incubation on ice, and centrifuged at 16,100 g at 4°C for 10 min. Supernatants were transferred to a new tube. 25 μl of each sample was collected and subjected to RNA extraction as \"input samples\".  Prior to the immunoprecipitation step, 100μl Protein A Dynabeads (Invitrogen) were washed 3 times with buffer A (10mM HEPES, 150mM KCl, 5mM MgCl2 and 1% NP40 (pH 7.4) before incubation with 4μg pS6 antibody against Ser240/244 (#2215, Cell Signalling Technology) and 0.1% bovine serum albumin (BSA) in buffer A (300 μl per IP sample) at 4°C overnight with continuous mixing on an end-over-end rotator. On the next day, the antibody-bead conjugates were washed twice with wash buffer D (10 mM of HEPES, 350 mM of KCl, 5 mM of MgCl2, 2 mM of DTT, 1% NP40, 100 U/mL of RNasin, 100mg/mL of cycloheximide, and Roche protease and phosphatase inhibitor cocktails). After the last wash, the beads were resuspended in 200 μl of homogenisation buffer C supplemented with 50 μl of 10% NP40 and 10 μl of DHPC for 1 mL of buffer C and 1 μM of ZK10 (a gift from Dr. Zackary Knight, UCSF, US). The remaining sample supernatant was added to the antibody-beads conjugates, resuspended by pipetting and mixed with a rotator at 4°C for 10 min. The beads were washed 4 times with 0.9 mL of ice-cold wash buffer D and resuspended in 350 μl of RLT buffer and allowed for 5 min incubation on ice. The supernatant was then collected as “IP sample.” RNA was extracted using the RNeasy Micro kit (Qiagen). RNA quantity was assessed using the Quant-iT RiboGreen RNA Assay kit (Thermo Fisher Scientific), and the samples' RNA quality was assessed using Pico chips on an Agilent Bioanalyser. The samples were cleared from DNA contamination using a TURBO DNA-free kit (Thermo Fisher Scientific). cDNA was prepared using a Smart Seq v4 Ultra Low Input RNA kit (Takara Clontech). Library preparation was done using Nextera XT Library Prepration kit (Illumina)