GSM3560257: K562_CTCF_150mM_(20190104_SH_Hs_C15_20181224,6); Homo sapiens; OTHER
Bench-top CUT&Tag: Cells were harvested, counted and centrifuged for 3 min at 600 x g at room temperature. Aliquots of up to 500,000 cells per final sample, were washed twice in 1.5 ml Wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1X Protease inhibitor cocktail) by gentle pipetting. Concanavalin A coated magnetic beads (Bangs Laboratories) were prepared as described [Skene, P.J. & Henikoff, S. Elife 6:e21856 (2017)] and 10 µl of activated beads were added per sample and incubated at RT for ~10 minutes. The unbound supernatant was removed and bead-bound cells were resuspended in 50-100 µl Dig-wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1X Protease inhibitor cocktail; 0.05% Digitonin) containing 2mM EDTA and a 1:50 dilution of the appropriate primary antibody. Primary antibody incubation was performed on a nutating platform for 2 hr at room temperature (RT) or up to several days at 4 oC. The primary antibody was removed by placing the tube on the magnet stand to clear and pulling off all of the liquid. To increase the number of Protein A binding sites for each bound antibody, an appropriate secondary antibody (such as Guinea Pig anti-Rabbit IgG antibody for a rabbit primary antibody) was diluted 1:50 in 50-100 µl of Dig-Wash buffer and cells were incubated at RT for 30 min. Cells were washed using the magnet stand 2-3x for 5 min in 1 ml Dig-Wash buffer to remove unbound antibodies. A 1:200 dilution of pA-Tn5 adapter complex (~0.04 µM) was prepared in Dig-med Buffer (0.05% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail). After removing the liquid on the magnet stand, 50-100 µL was added to the cells with gentle vortexing, which was incubated with pA-Tn5 at RT for 1 hr. Cells were washed 2-3x in 1 ml Dig-med Buffer to remove unbound pA-Tn5 protein. Next, cells were resuspended in 90 µL Dig-med Buffer and 10 µL 100 mM MgCl2 was added with gentle vortexing, and incubated at 37 for 1h. To stop tagmentation, 45 µL 0.5M EDTA, 5.5 µL 10% SDS and 1 µL 20 mg/ml Proteinase K was added, and tubes were incubated at 55 oC for 30 min and then at 70 oC for 20 min to inactivate Proteinase K. A volume of 122 µL Ampure XP beads was added, vortexed, incubated at RT with beads for 15 min at RT, washed twice gently in 80% ethanol, and eluted in 30 µL 10 mM Tris pH 8.0. To make libraries, 21 µL DNA was mixed with 2 µL of a universal i5 and a uniquely barcoded i7 primer [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)], using a different barcode for each sample. A volume of 25 µL NEBNext HiFi 2x PCR Master mix was added and mixed. The sample was placed in Thermocycler with heated lid using the following cycling conditions: 72 °C for 5 min (gap filling); 98 °C for 30 sec; 14 cycles of 98 °C for 10 sec and 63 °C for 30 sec; final extension at 72°C for 1 min and hold at 8 oC. Post-PCR clean-up was performed by adding 1.1X volume of Ampure XP beads (Beckman Counter), and libraries were incubated with beads for 15 min at RT, washed twice gently in 80% ethanol, and eluted in 30 µL 10 mM Tris pH 8.0.
GEO Accession
GSM3560257