GSM8991944: NL2; Mus musculus; OTHER
Frozen tissues were homogenized with ice-cold Dounce homogenizer in 4 ml of freshly made low sucrose buffer (LSB) [15 mM Tris-HCl (pH 7.4, Invitrogen), 25 mM KCl (Fisher scientific), 5 mM MgCl2 (Fisher Scientific), 1 mM DTT (Sigma-Aldrich), 1 tablet / 20 ml of EDTA-free proteinase inhibitors (Thermo Scientific), 0.5 mM  Spermidine (Sigma-Aldrich), 0.15 mM Spermine (Sigma-Aldrich), 250 mM Sucrose (Sigma-Aldrich)]. After the homogenization, Nonidet P-40 (Roche) was added to make final 0.1% Nonidet P-40, and mix. The homogenates were filtered through a 70 µm filter, followed by a 40 µm filter. The filtered homogenates were transferred to 2 tubes and centrifuged at 600×g, 4 degrees for 10 min, then the pellet was resuspended in 0.5 ml (per tube) of wash buffer (WB) [15 mM Tris-HCl (pH 7.4, Invitrogen), 25 mM KCl (Fisher scientific), 5 mM MgCl2 (Fisher Scientific), 1 mM DTT (Sigma-Aldrich), 1 tablet / 20 ml of EDTA-free proteinase inhibitors (Thermo Scientific), 0.5 mM  Spermidine (Sigma-Aldrich), 0.15 mM Spermine (Sigma-Aldrich), 250 mM Sucrose (Sigma-Aldrich), 0.1% Nonidet P-40]. Add 0.9 ml (per tube) of Sucrose Cushion Buffer [15 mM Tris-HCl (pH 7.4, Invitrogen), 25 mM KCl (Fisher scientific), 5 mM MgCl2 (Fisher Scientific), 1 mM DTT (Sigma-Aldrich), 1 tablet / 20 ml of EDTA-free proteinase inhibitors (Thermo Scientific), 0.5 mM  Spermidine (Sigma-Aldrich), 0.15 mM Spermine (Sigma-Aldrich), 1.2 M Sucrose (Sigma-Aldrich)] to the suspension, and gently mix. Carefully overlay this mixture (1.4 ml) on top of the 0.5 ml of Sucrose Cushion Buffer, and centrifuged at 13,000×g, 4 degrees for 45 min. Discard the supernatant, and the pellet was resuspended in total 1 ml of WB. Washed and resuspended GFP-Trap® Magnetic Particles M-270 (Protein Tech) were added, and the mixture were incubated at 4 degrees for 1 hour with continuous rotation. After the removal of the supernatant, the magnetic beads were washed in WB, for three times. After removing the WB, CUT&RUN was performed as described in the user manual (Cell Signaling, 86652S), with UTX (D3Q1I) Rabbit mAb (Cell Signaling, 33510S) and Rabbit (DA1E) mAb IgG XP Isotype Control (Cell Signaling, 66362). Sequence library was made using Zymo-Seq™ SPLAT DNA Library Kit (Zymo Research).