GSM9177923: CD201+CD150+KSL wild-type cultured HSC CUT&Tag H3K4me3; Mus musculus; OTHER
FACS sorted cells were pelleted at 300 x g for 10 mins at RT before resuspending in 1 ml NE1 buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton X-100, 20% glycerol, 1× Complete Protease Inhibitor [EDTA-free]) and incubating on ice for 10 mins. Nuclei were pelleted at 500 x g for 10 mins at 4 C before resuspending in 0.1% formaldehyde and incubating at RT for 2 mins, followed by quenching with 78 mM Glycine and pelleting the nuclei at 500 x g for 10 mins at 4°C. Nuclei were resuspended in Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× Roche Complete Protease Inhibitor [EDTA-free]) and counted by trypan blue staining. Concanavalin A (Con A) beads were activated by washing (2x) with 1 ml Concanavalin Activation Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂). For each antibody/sample combination, 5 ul of activated ConA beads were added to 5x104 . Nuclei were incubated with 0.5 ul of primary antibody (antibodies listed in Supplementary Table 3) in 25 ul of antibody buffer (0.1% BSA, 2 mM EDTA in Wash Buffer) for either 1 hr at RT or overnight at 4oC. Nuclei were then incubated with 25 ul of 1:100 secondary antibody (typically Guinea Pig anti-Rabbit IgG) for 30 mins at RT. Nuclei were washed with 200 ul Wash Buffer and incubated with 25 ul 1:20 pAG-Tn5 (EpiCypher) in WB-300 (300 mM NaCl in Wash Buffer) for 1hr at RT. Nuclei were washed (x2) with 200 ul of WB-300 to remove unbound pAG-Tn5 and then incubated at 37oC for 1hr in 50 ul Tagmentation buffer (10 mM MgCl₂ in WB-300). Tagmentation was halted by washing with 50 ul TAPS Wash Buffer (10 mM TAPS pH 8.5, 0.2 mM EDTA) before lysing the nuclei with 5 ul SDS-ProK Buffer (1% SDS, 10 mM TAPS pH 8.5, Thermolabile Proteinase K) and incubating for 1 hr at 37oC and 1hr at 58oC. SDS was neutralised with 15 ul 6% Triton and tagmented DNA amplified with NEBNext Ultra II Q5 Master Mix and indexed primers (thermal profile: 58 °C 5 min, 72 °C 5 min, 98 °C 5 min, [98 °C 10 sec, 60 °C 10 sec, 72 °C 1 min] ×12). Libraries were cleaned with Agencourt AMPure XP beads. Libraries were cleaned with Agencourt AMPure XP beads.