title: GSM7698683: AgRP neurons, fasted, RNA, rep3; Mus musculus; RNA-Seq
library_construction_protocol: TRAP IP was performed for each sample thus: 4 frozen ARC tissue samples from NuTRAPAgRP mice between 6 and 11 weeks of age were pooled and lysed with Dounce homogenizers in low-salt IP buffer (50mM Tris [pH7.5], 12mM MgCl2, 100mM KCl, 1% NP-40; 100 µg/ml cycloheximide, 2mM DTT, 0.2units/ml RNasin, 1x Roche Complete EDTA-free protease inhibitor). After centrifugation, the supernatant was incubated with GFP ab290 antibody (Abcam) on a rotor, then incubated again with Dynabeads Protein G (Thermo Fisher). RNase-inhibitors were used throughout the protocol to mitigate potential RNA degradation. RNA was isolated with the RNeasy Micro Kit (Qiagen) according to manufacturer's instructions, and then reverse-transcribed and analyzed by qRT-PCR as described above. TRAP-isolated RNA (<100ng) was treated with the Ribo-Zero rRNA removal kit (Epicentre) to deplete ribosomal RNA and converted into double stranded cDNA using NEBNext mRNA Second Strand Synthesis Module (E6111L). cDNA was subsequently tagmented and amplified for 12 cycles by using Nextera XT DNA Library Preparation Kit (Illumina FC-131). Sequencing libraries were analyzed by Qubit and Agilent Bioanalyzer, pooled at a final concentration of 12pM, and sequenced on a NextSeq500. AgRP neuronal nuclei were isolated from dissected ARCs from 6-11-week-old mice and were snap frozen and stored at -80C. Isolated ARCs were dounce homogenized in nuclear preparation buffer (NPB; 10 mM HEPES [pH 7.5], 1.5mM MgCl2, 10 mM KCl, 250 mM sucrose, 0.1% NP-40, and 0.2 mM DTT), and homogenates were filtered through a 100 μM strainer and centrifuged to pellet the nuclei. Nuclei were washed with NPB, re-suspended in nuclear sorting buffer (10 mM Tris [pH 7.5], 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.1% NP-40, 0.2 mM DTT), and filtered again through a 40 μM strainer. Isolated nuclei were sorted by flow cytometry based on AgRP neuron-specific GFP expression. With this approach we routinely obtain ~2000 AgRP neuronal nuclei per mouse. Nuclei from 4 pooled mice (per sample), were isolated, subjected to FANS, and 10,000 nuclei were collected into 500-750uL PBS (0.1% NP40) in 1.5 mL microcentrifuge tubes and stored on ice. Tubes were spun at 1000 rpm for 10 mins, at 4°C. Supernatant was removed via gentle decantation. Nuclei pellets were subjected to tagmentation after resuspension in 50ul of transposase reaction mix: 25 ul TD buffer, 2.5 ul TN5 transposase, 22.5 ul dH20. Nuclei were incubated at 37°C with shaking at 600 rpm for 30 mins, then placed on ice. DNA was purified using a Qiagen minelute PCR purification kit and eluted in 10 ul of Qiagen Elution Buffer Library construction was conducted using an initial PCR amplification using 10 ul of transposed DNA, 11ul H2O, 2ul of 25uM Primer 1, 2ul of Primer 2, and 25 ul of NEBNext HF 2X PCR mix. The following initial amplification cycle was used: 1 cycle or 72ºC (5min), 98ºC (30s), 5 cycles of 98ºC (10s), 63C (30s), 72ºC (1 min), hold at 4ºC. A side PCR reaction was conducted to determine the optimal number of amplification cycles used during the library construction, as previously described50,  using 5 ul PCR amplified DNA, 4.41 ul H2O, 0.25ul Primer 1 Adaptor, 0.25ul Primer 2 Adaptor 2 (barcoded), 0.09 ul 100x SYBR Green (Diluted from 10,000X stock with H2O), 5 ul NEBNext HF 2X PCR mix. The following initial amplification cycle was used: 1 cycle of 98ºC (30s), 20 cycles of 98ºC (10s), 63ºC (30s), 72ºC (1 min). Amplification curves were assessed to determine the number of additional cycles needed during library construction50. The requested additional number of cycles was completed by adding the sample back to the thermocycler in its same tube and master mix. Amplified libraries were purified with a Qiagen Minlute PCR purification kit, eluted in 20ul, and the volume was brought up to 100 ul with additional EB. The eluent was subjected to a two-phase size selection using Ampure Beads, initially .55X volume of elutant (55ul) was used, followed by 1.5X volume of initial sample volume (100ul x 1.5 = 150ul) minus the existing PEG that is already in the sample (150ul – 55ul = 95 ul). Finally, the library was eluted using 50ul of EB. Libraries were sequenced on a Next-seq 500 (Illumina) ~4,000 ARC nuclei, from two mice, were isolated and subjected to FANS, and collected into 0.75 mL of C&R Wash Buffer (low salt) buffer (20 mM HEPES-KOH [pH 7.5], 75 mM NaCl, 0.5 mM Spermidine, 0.1% BSA and 1x protease inhibitor cocktail from Sigma). 180 μL BioMagPlus Concanavalin A beads slurry was washed twice and re-suspended in 180 μL Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2), and added to the nuclear suspension. The mixture was thoroughly mixed on a rocker for 10 minutes to allow binding of nuclei to the beads. The nuclei were pelleted with a magnet stand and blocked with Wash Buffer (20 mM HEPES-NaOH pH 7.5, 75 mM NaCl, 0.5 mM Spermidine, 0.1% BSA and 1x protease inhibitor cocktail from Sigma) supplemented with 2 mM EDTA. After washing once with Wash Buffer, nuclei were re-suspended in 50 μL Wash Buffer containing 2 μg of anti-IRF3 antibody (Proteintech, 11312-1-AP), and incubated overnight to allow antibody binding. The next day, nuclei were washed once on a magnet stand with Wash Buffer and incubated with 1:1000 pA-MN in 200 μL Wash Buffer. After 1 hour, nuclei were washed once to remove unbound pA-MN, re-suspended in 150 μL Wash Buffer, and then chilled on a 0°C metal block in a water-ice mixture. 3 μL of 100 mM CaCl2 was added to activate pA-MN, and the mixture was incubated at 0°C for 60 min. The reaction was stopped by adding 150 μL 2XSTOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A and 40 μg/mL glycogen). The protein-DNA complexes were released by centrifugation and then digested by proteinase K at 50°C overnight. DNA was extracted by ethanol precipitation, followed by Qubit fluorometer and bioanalyzer quality control. CTCF optimization experiments were performed using DAPI-treated nuclei isolated from whole mouse cerebellum, and a CTCF antibody from (Millipore, 07-729). Peri-hypothalamus IRF3-2D IgG (IgG C&R, Figures 6, S6E, S6F) sample was generated by stereotaxically injecting a IRF3LSL-2D mouse with a AAV2-Cre-GFP in the peri-hypothalmus and ~200K GFP+ nuclei were isolated by FANS and used for CUT&RUN on the same day the IRF3Ag C&R sample was generated. IRF3 CUT&RUN from hepatocytes was conducted using primary hepatocytes isolated from IRF3LSL-2D mice as described, and transiently transduced ex vivo with adenovirus expressing Cre recombinase (Ad-Cre). IRF3 and IgG CUT&RUN from immortalized AgRP neurons (IRF3 C&R AgRPin vitro and IgG C&R AgRPin vitro, Figures S6E, S6F) was conducted using those cells transiently transduced with Ad-CMV-IRF3-2D-hPGK-eGFP (Vector Builder). CUT&RUN libraries were prepared using the NEB Ultra II library preparation kit with important modifications35. Briefly, less than 30 ng DNA was used as input. End prep was performed at 20°C for 30 minutes and then 50°C for 60 minutes, as reduced temperature prevents melting of short DNA fragments (Liu et al., 2018). 5 pmol adaptor was added and ligated to end prep products at 20°C for 15 minutes. USERTM enzyme was then used to cleave the uracil in the loop. 1.7x volume of AMPure beads were used to purify the ligation product, which aids in the recovery of short fragments. To amplify the library, the ligation product was mixed with 2x Ultra II Q5 mix, universal primer and index primers. PCR was carried out as follows: 98°C 30 seconds, 12 cycles of 98°C 10 seconds and 65°C 10 seconds, and final extension 65°C 5 minutes. The PCR product was subjected to double size selection with 0.8x and then 1.2x AMPure beads. The purified library was quantified with Qubit and Tapestation. Libraries were pooled at similar molar amounts and sequenced using Nextseq500 platform.