title: GSM8666749: Brain, sleep deprived, ZT6, rep3, IgG; Mus musculus; OTHER
library_construction_protocol: For the brain sample collection, 6 hours sleep deprivations during ZT0 – ZT6 was performed by a gentle handling after 2 weeks of acclimatization. The samples were flash-frozen in liquid nitrogen and stored at -80°C until use. Intact nuclei were extracted used for CUT&RUN. Minced cerebral cortex tissues were incubated in the Nuclear Extraction Buffer: 20mM HEPES (Gibco, 15630080), 10mM Potassium chloride (Sigma-Aldrich, P9541), 20% (v/v) Glycerol (Sigma-Aldrich, G5516), 0.1% NP-40 (Thermo Scientific, 85124), 0.5mM Spermidine (Sigma-Aldrich, S0266), 1X Halt Protease Inhibitor Cocktail (Fisher Scientific, 78425) and 2% Bovine Serum Albumin (Sigma-Aldrich, B6917), for 10min on ice and filtered through 40µm CellPro Premium Cell Strainer (DOT SCIENTIFIC INC., CS4040). These steps were repeated three times before proceeding the following workflows. CUT&RUN wes performed according to the manufacture's instruction. In brief, activated Concanavalin A Conjugated Paramagnetic Beads (: ConA beads, EpiCypher, 211401) were incubated with the extracted nuclei to bind for 10min at room temperature. The ConA beads-nuclei mixture was resuspended in cold Antibody Buffer: 20mM HEPES (Gibco), 150mM Sodium chloride (Sigma-Aldrich, S5150), 0.5mM Spermidine (Sigma-Aldrich), 1X Halt Protease Inhibitor Cocktail (Fisher Scientific), 0.05% Digitonin (Millipore Sigma, 300410), 2mM EDTA (Invitrogen, 15575020) and 2% Bovine Serum Albumin (Sigma-Aldrich), and incubated with 0.5µg of anti-HSF1 antibody (Cell Signaling, 4356S), SNAP-ChIP Certified H3K4me3 Positive Control Antibody (Thermo Fisher, MA511198) or Rabbit IgG Negative Control Antibody (EpiCypher, 130042) per 5x105 nuclei at 4°C overnight on nutator. After washing twice in cold Digitonin Buffer: 20mM HEPES (Gibco), 150mM Sodium chloride (Sigma-Aldrich, S5150), 0.5mM Spermidine (Sigma-Aldrich), 1X Halt Protease Inhibitor Cocktail (Fisher Scientific), 0.05% Digitonin (Millipore Sigma, 300410) and 2% Bovine Serum Albumin (Sigma-Aldrich), the ConA beads-nuclei-antibody mixtures were resuspended in Digitonin Buffer and incubated with 2.5µL of CUTANA pAG-MNase (EpiCypher,151116) per 5x105 nuclei for 1 hour at 4°C on nutator for binding. The mixtures were washed twice, resuspended in Digitonin Buffer with 2mM Calcium chloride (Sigma-Aldrich, 21115) and incubated at 4°C for 2 hours for chromatin digestion. After the incubation, Stop Buffer: 340mM Sodium chloride (Sigma-Aldrich), 20mM EDTA (Invitrogen), 4mM EGTA (Sigma-Aldrich, E8145), 50µg/mL RNase A (Thermo Scientific, EN0531), 50µg/mL Glycogen (Roche, 10901393001), 0.5mM Spermidine (Sigma-Aldrich), 1X Halt Protease Inhibitor Cocktail (Fisher Scientific), 0.02% Digitonin (Millipore Sigma, 300410) and 2% Bovine Serum Albumin (Sigma-Aldrich), was added and incubated for 10min at 37°C to stop the digestion. The cleaved chromatins were collected from the supernatant and purified using Monarch PCR&DNA Cleanup Kit (BioLabs, T1030L) according to the manufacture's instruction. Sequencing libraries for each condition were prepared using 10ng of CUT&RUN enriched DNA fragments in total made by pooling equal amount DNAs of three replicates. End repair was performed using NEBNext Ultra II End Repair/dA-Tailing Module (New England BioLabs, E7546L) according to the manufacture's instructions. Then, the samples were ligated to the NEBNext Adaptors (New England BioLabs, E6440L) and cleaned up using 1.75X (v/v) SPRIselect beads (Beckman Coulter, B23318). Adaptor-ligated DNA fragments were amplified using NEBNext Unique Dual Index Primer Pairs (New England BioLabs, E6441A) with NEBNext Ultra II Q5 Master Mix (New England BioLabs, M0544S). PCR was performed using the following parameters: initial denaturation at 98°C for 30sec, 14 cycles of denaturation at 98°C for 10sec and annealing/extension at 65°C for 75sec, final extension at 65°C for 5min. Size selections for 150-500bp were performed on the PCR products by applying 0.6X and 1.2X SPRIselect beads successively.