GSM9549700: Agrp MFF KO IP 3; Mus musculus; RNA-Seq
Mice were sacrificed at 11-12 weeks of age in a random-fed or overnight fasting state by decapitation. Brains were rapidly removed from the skull and placed in a brain slicer blocker (World Precision Instruments) with the inferior view upwards. A 3 mm coronal slice was obtained using two blades. The first blade was located at the intersection between the brainstem and the midbrain, and the second one was placed 3 mm forward, containing the ARC. Then, the brain slice was placed on a plate covered with aluminum foil on ice. Two parasagittal cuts were made at 1 mm distance from the 3V, followed by a horizontal cut just above the ARC. Once the ARC was isolated, it was frozen in liquid nitrogen and stored at -80ºC until further processing. For the Translating Ribosome Affinity Purification (TRAP) protocol 3 bacTRAP control (Mffwt/wt;AgRP-IRES-Cretg/wt;RpL10a-GFPfl/wt) and 3 experimental (Mfffl/fl;AgRP-IRES-Cretg/wt;RpL10a-GFPfl/wt) mice (mixed genders) were pooled for each replicate, accounting for three to four replicates per group. Protein A Dynabeads (Thermo Fischer Scientific) were washed three times in 0.15 M KCl IP wash buffer (20 mM HEPES pH 7.4; 5 mM MgCl2; 150 mM KCl; 1% NP40; 0.5 mM DTT and 100 mg/ml cycloheximide). Beads were resuspended in 275 µl 0.15M KCl IP wash buffer, loaded with 50 µg of 19C8 and 50 µg of 19F7 (Memorial Sloan Kettering Monoclonal Antibody Facility) and incubated with slow end-over-end mixing at 4ºC overnight. Pooled hypothalamic tissue was homogenized in 1 ml of lysis buffer (20 mM HEPES pH 7.4; 5 mM MgCl2; 150 mM KCl; 0.5 mM DTT; 40 U/ml RNasin; 100mg/ml cyclo-heximide; protease and phosphatase inhibitor cocktails containing 1 tablet of cOmplete mini EDTA-free protease inhibitor cocktail/7 ml and 2 tablets of PhosSTOP/10 ml) two times at 250 rpm, and nine times at 750 rpm on a rotating glass/teflon potter homogenizer at 4ºC. Homogenates were transferred to low-binding microcentrifuge tubes (Nonstick, RNase-free microfuge tubes, 1.5 ml; Ambion; Thermo Fischer Scientific) and centrifuged at 2000g for 10 min at 4ºC. The supernatant was transferred to a new tube on ice, and 1/9 sample volume 10% NP-40 (final concentration: 1%) and 1/9 sample volume 300 mM DHPC (final concentration: 30 mM) were added. This solution was mixed, incubated on ice for 5 min, and then centrifuged at 20000g for 10 min at 4ºC. Next, the resulting supernatant was transferred to a new tube, and a 30 µl aliquot was removed, transferred to a new tube, frozen in liquid nitrogen and stored at -80ºC for purification as input RNA. The remaining supernatant was incubated with 200 µl of antibody-bound beads (800 – 1000 µl SN) at 4ºC for 1 h with end-over-end mixing. Then, beads were collected with a magnet in an ice bucket 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 mg/ml cycloheximide). This step was repeated three more times in order to wash the beads. After the final wash, the beads were collected with a magnet, and the SN was removed. In order to elute the RNA, RLT buffer (350 µl) (RNeasy Micro Kit, Qiagen) was added to the beads and incubated at RT for 5 min. Finally, RNA was purified using the RNeasy Micro Kit (Qiagen), and its integrity was assessed using an Agilent 2100 bioanalyzer. Pre-amplification was performed using the Ovation RNA-seq system (V2). Total RNA was used for first-strand cDNA synthesis, using both poly(T) and random primers, followed by second-strand synthesis and isothermal strand-displacement amplification. For library preparation, the Illumina Nextera XT DNA sample preparation protocol was used, with 1 ng cDNA input.