GSM6306785: SCARB2ko2; Homo sapiens; OTHER
GSM6306785
Cells were harvested, counted and were washed with Wash Buffer. Concanavalin A Beads were prepared with Binding buffer and 10 µL of activated beads were added per sample, invert to mix and incubate at room temperature. The primary antibody incubation was performed on a rotating platform overnight at 4 °C. Beads were washed and incubated with secondary antibody. After incubation, the beads were washed and incubated with hyperactive pA/G-transposon (pA/G-Tnp) at RT. Next, cells were re-suspended in Trueprep Tagment Buffer L and the DNA fragments were extracted with DNA Extract Beads. Beads were washed twice with Buffer WA and Buffer WB. After drying ~ 5 min, 20 µL ddH2O was added. To amplify libraries, 15 µL DNA was mixed with 25 µL 2×CUT&Tag amplification mix, 5 µL N5, and 5 µL N7 primer, using a different barcode for each sample. The sample was placed in a Thermocycler with a heated lid using the following cycling conditions: 72 °C for 3 min; 95 °C for 30 min; 14 cycles of 98 °C for 10 s and 60 °C for 5 s; final extension at 72 °C for 1 min and hold at 4 °C. Post-PCR clean-up was performed by adding 100 µL VAHTS DNA Clean Beads (Vazyme, #N411), and libraries were incubated with beads for 5 min at RT, washed twice gently in 80% ethanol, and eluted in 20 µL ddH2O.