GSM5605742: IR expressed in tancytes of NCD-fed animals IP1; Mus musculus; RNA-Seq
Arcuate and median eminence regions were collected, frozen on dry ice and stored at -80°C.  Four to seven hypothalami were pooled per replicate. Further steps of the immunopurification of L10a-GFP tagged ribosomes and subsequent extraction of RNA were performed exactly as previously described94,96.  Protein A Dynabeads (375 µl per IP, Invitrogen) were washed three times (1mL) in 0.15M KCl IP wash buffer (20mM HEPES [pH 7.4], 5 mM MgCl2, 150 mM KCl, 1% NP40, 0.5 mM DTT and 100 mg/ml cycloheximide, resuspended in 275 µl 0.15M KCl IP wash buffer with 50 mg of 19C8 and 50 mg of 19F7 (Cat# Htz-GFP-19F7, RRID:AB_2716736 and Htz-GFP-19C8, RRID:AB_2716737, Memorial Sloan Kettering Monoclonal An-tibody Facility) and incubated with slow end-over-end mixing at 4°C overnight. Next day, beadswere washed three times (1 mL) in 0.15M KCl IP wash buffer to remove unbound anti-GFP antibody. Pooled hypothalami were 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 mg/ml cycloheximide, protease and phosphatase inhibitor cocktails [1 tablet of cOmplete mini EDTA-free protease inhibitor cocktail/7 mL and 2 tablets of PhosS-TOP/10 ml]) using a handheld homogenization device at 4°C for 30 s. Homoge-nates were centrifuged at 2,000x g for 10 min at 4°C. The supernatant was trans-ferred to a new tube on ice, and 1/9 sample volume of  10% NP-40 (Applichem) and 300 mM DHPC (Avanti Polar Lipids) with final concentration 1% and 30 mM, re-spectively, incubated on ice for 2 min and then centrifuged at 17,000 x g for 10 min at 4°C. The resulting supernatant was transferred to a new tube, and a 30 µl aliquot of the supernatant was flash frozen separately on dry ice for purification as input RNA. Then, 200 µl of anti-GFP-coated  beads were added (800 -1000 ml superna-tant) and incubated at 4°C for one hour with end-over-end mixing. Beads were col-lected with a magnet and resuspended in 1000 µl of 0.35 M KCl IP wash buffer (20 mM HEPES [pH 7.4], 5 mM MgCl2, 0.35 M KCl, 1% NP40, 0.5 mM DTT, 100 mg/ml cycloheximide). Beads were collected with MagnaRack (Invitrogen) and washed three times (1mL) with  0.35 M KCl IP wash buffer. The collected beads were incu-bated in  RLT buffer (350 µL at RT for 5 minutes. Input and IP RNA were isolated and purified subsequently using the Rneasy Micro Kit (QIAGEN). RNA was eluted in 10 µL in nuclease free water. RNA integrity was assessed using an Agilent 2100 bioanalyzer and RNA concentration was measured using Qubit fluorometer (Ther-mofisher Scientific). Due to low amount of input material, pre-amplification using the Ovation RNASeq System V2 was performed as previously described. 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. After validation (Agilent 2200 TapeStation) and quantification (Invitrogen Qubit System), transcriptome libraries were pooled. The pool was quantified using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection and sequenced on an Illumina NovaSeq sequencing instrument with a 2 x 100 bp paired-end read length.