GSM8833588: iAT1_CUT1_H3K27me3; Homo sapiens; OTHER
For the isolation of iAT2 and iAT1 cells, the cells cultured on micro-patterned plates were first stained with Hoechst-33342 for 30 minutes at 37°C, followed by incubation with Accutase at 37°C for 20 minutes and dissociation by gentle pipetting. For the isolation of iATCs, the cells were incubated with Accutase, gently dissociated by pipetting, and stained with an APC-conjugated anti-human CD54 antibody for 20 minutes at 4°C. Each positive cell population was then sorted using fluorescence-activated cell sorting. To make the CUT&Tag libraries, the dissociated cells were washed with PBS supplemented with 1X Protease Inhibitor (Roche, # 11873580001), and subsequently washed with wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.25 mM spermidine, 1X Protease Inhibitor (Roche, # 11873580001)). Then, we resuspended 50,000 cells in 500 µl of the wash buffer supplemented with 5 mM sodium butyrate. We added 2 µl of the concanavalin A-conjugated magnetic beads to the cell suspension. Next, after magnetic separation of the beads and removal of the supernatant, we resuspended the beads in 50 µL of ice-cold wash buffer, supplemented with 5 mM sodium butyrate, 0.05% digitonin, 2 mM EDTA, and 0.1% BSA together with 0.5 µl primary antibody against H3K4me3 (MAB Institute, MABI0304) and H3K27me3 (MAB Institute, MABI0323). After rotating incubation at room temperature for 3 hours, the buffer containing the primary antibody was replaced with 100 µl of ice-cold wash buffer, supplemented with 5 mM sodium butyrate, 0.05% digitonin, together with 1 µl secondary rabbit anti-mouse IgG antibody (Abcam, #ab46540), and was incubated at 4 °C for overnight. After washing the beads with Dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, 1X Protease Inhibitor (Roche, # 11873580001), 5 mM sodium butyrate) twice, we applied 100 µl of the Dig-300 buffer containing 3.6 femtomol pA-Tn5 transposome and incubated the mixture for 1 hour at room temperature. After washing the beads with the Dig-300 buffer twice, we resuspended the beads in 50 µl of the Dig-300 buffer supplemented with 10 mM MgCl2 and incubated them at 37 °C for 1 hour. We then removed the supernatant and resuspended the beads in 20 µl of 10 mM TAPS buffer (pH 8.5) together with 0.5 µl thermolabile proteinase K (NEB, #P8111S), and incubated the mixture at 37 °C for 30 minutes, followed by incubation at 55 °C for 20 minutes to inactivate the proteinase. We directly performed PCR-amplification of the library from the tube after adding the reaction mixture (KAPA BIOSYSTEMS, #KK2602) using index primers. The amplified libraries were purified with 1.3 volumes of AMPure XP beads.