title: GSM7246555: NPC, shSOX2, H3K4me1, biol rep1; Homo sapiens; OTHER
library_construction_protocol: Profiling of histone markers in SH-SY5Y cells was performed with CUTANA™ CUT&Tag kit (Epicypher, cat#14-1101) following the manufacturer's protocol. Briefly, desired Concanavalin A (ConA) coated magnetic beads (Bangs Laboratories, cat#BP531) for all planned CUT&Tag reactions (11 μL per reaction) were processed together and activated in cold Bead Activation Buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). 10 ul of activated ConA beads were aliquoted into each 8-strip PCR tubes for individual reactions and kept cold until needed. WT and INTS10 KO SH-SY5Y cells were harvest, counted and centrifuged for 3 min at 600⨉g at room temperature in two individual 1.5 mL tubes. Total desired amount of WT and INTS10 KO cells (200, 000 cells per experiment) were washed once with PBS at room temperature. To isolate nuclei, cell pellets were resuspended in cold Nulcear Extraction buffer (20 mM HEPES–KOH, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine, 1 x Protease inhibitor), incubated for 10 min on ice and centrifuged for 3 minutes at 600⨉g at 4 °C to remove the supernatant. Fluffy nuclei pellets were resuspended in 100 μL/reaction cold NE buffer and aliquoted into 8-strip PCR tubes containing 10 μL of activated ConA beads. Nuclei-beads slurry were vortex gently to mix and incubated for 10 minutes at room temperature. 50 μL cold Antibody150 Buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, 2 mM EDTA, 1X protease inhibitor ) containing 0.5 ug of H3K4me1 or H3K27ac antibody was added immediately to the beads after the removal of the supernant and vortex gently and thoroughly. The reactions were incubated on nutator overnight at 4 °C . On the next day, anti-rabbit secondary antibody master mix was prepared first by diluting secondary antibodies in cold Digitonin 150 buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, 1X Protease inhibitor). After removing the supernatant from the beads slurry, 50 μL/reaction cold secondary antibody master mix was added to each reaction and mixed well by gentle vortexing. The reactions were incubated on nutator for 30 minutes at room temperature. After two washes with cold Digitonin 150 buffer (), 50 μL/reaction cold Digitonin 300 buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1 X protease inhibitor, 0.01% Digitonin ) containing 2.5 μL pAG-Tn5 was added to the beads slurry. Beads were thoroughly resuspened by gentle vortexing and incubated on nutator for 1 hour at room temperature. After two washes with 200 μL/reaction cold Digitonin 300 buffer, 50 μL/reaction cold Tagmentation buffer (Digitonin300 Buffer, 10 mM MgCl2 ) was added to each reaction and the tubes were incubated for 1 hour at 37 °C in a thermocycle. 50 μL/reaction RT TAPS buffer (10 mM TAPS, pH 8.5, 0.2 mM EDTA) was then used to wash the reactions and 5 μL/reaction RT SDS Release buffer (10 mM TAPS, pH 8.5, 0.1% SDS) was added to quench the tagmentation reaction. 15 μL/reaction RT SDS Quench buffer (0.67% Triton-X 100 in Molecular grade H2O) was then used to neutralize SDS, which inhibits PCR. Library amplification was performed directly on the entire reaction mixture by adding dual primers and CUTANATM High Fidelity 2x PCR Master Mix. After PCR, the library DNA was purified using AMPure XP beads (ThermoFisher, cat#A63881) and quantified by Qubit. CUT&TAG (Kaya-Okur et al. 2020)