GSM9890039: TX-Maged1-2tag Xist-B6 Rep3; Mus musculus; OTHER
Cells were dissociated with Accutase for 5 mins, followed by media addition to stop dissociation. They were sorted with a BD FACS Aria II flow cytometer (BD Biosciences) with a 100μm nozzle and a 5B-3YG-2R-1DB laser configuration for mScarlet-I3 (615/20) and mStaygold (510/20). The sideward and forward scatter areas were used for live cell gating. The height and width of the forward scatter and subsequently the height and width of the sideward scatter were used for singlet/doublet differentiation. Sorted cells were centrifuged, resuspended in cell culture medium andcounted. 10^5 cells were collected per CUT&Tag reaction and washed with 1mL wash buffer (20 mM HEPES–KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 10 mM sodium butyrate, protease Inhibitor and 1 mM PMSF). After washing, cells were resuspended in 100μL wash buffer. For every 10^5 cells, 10μL of BioMag Plus Concanavalin A beads were used, after being equilibrated in excess of binding buffer (20 mM HEPES–KOH pH 7.5,10 mM KCl,1 mM CaCl2, 1 mM MnCl2) and concentrated in 10uL of binding buffer. The cells were incubated with Concanavalin A beads by rotating for 10 min at room temperature. The beads with the bound cells were separated with a magnet and resuspended in 100 μl of ice-cold antibody buffer (wash buffer with 0.05% digitonin and 2mM EDTA). 1 μl of primary antibody (H3K9me3, Active Motif #39161) was added and beads were incubated on a rotator for 3 h at 4 °C. The beads were magnetically separated and resuspended in 100 μl of ice-cold Dig-wash buffer (wash buffer with 0.05% digitonin) and 1 μl of secondary antibody (Guinea pig anti-rabbit, antibodies online ABIN101961) and incubated for 1 h at 4 °C on a rotator. Next, the beads were washed three times with ice-cold Dig-wash buffer, and resuspended in 100 μl ice-cold Dig-300 buffer (20 mM HEPES–KOH pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, 10 mM sodium butyrate and 1 mM PMSF and protease inhibitor) with 0.4 μl 3×FLAG–pA-Tn5 (derived from Addgene 124601) preloaded with mosaic-end adaptors. Beads were incubated for 1 h at 4 °C on a rotator, and then washed four times with ice-cold Dig-300 buffer. While on ice, beads were resuspended in 50uL of tagmentation buffer (Dig-300 buffer, 10 mM MgCl2 and spike-in DNA). Spike-in DNA was prepared with PCR amplification of the Ampicillin resistance gene (AmpR) that also contains i5 and i7 primer binding sites on 5′ and 3′ ends, respectively. Tagmentation was carried out for 1 h at 37 °C. To stop tagmentation and solubilize DNA fragments, 2.25 μl of 0.5 M EDTA, 2.75 μl of 10% SDS and 0.5 μl of 20 mg/mL proteinase K were added, followed by vortexing for 5 s. Samples were incubated overnight at 55 °C followed by 30 min at 70 °C to inactivate proteinase K. The DNA fragments were purified with the ChIP DNA clean and concentrator kit (Zymo Research) and eluted in 25 μl of elution buffer. Libraries for sequencing were prepared by amplifying the purified fragments with indexed primers and NEBNext HiFi 2× PCR Master Mix for 14 amplification cycles. Post-PCR cleanup was performed with AMPure XP beads (1x volume), and the final libraries were eluted in 27 µl of 10 mM Tris (pH 8.0). Libraries were sequenced PE150.