GSM9834067: CUTandTAG, RPB1, aux2, AlFx; Homo sapiens; OTHER
Cells were crosslinked with 0.3% formaldehyde for 2 min, quenched with 225 mM glycine for 5 min. The crosslinked cells were washed once with cold PBS and resuspended in wash buffer [20 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1× cOmplete EDTA-free (Roche)]. Concanavalin A–conjugated magnetic beads were prepared by incubating Dynabeads MyOne T1 (Invitrogen) with biotinylated concanavalin A (Type IV; Merck Sigma-Aldrich) in bead wash buffer [PBS (pH 6.8) containing 0.01% Tween-20]44. Cells were immobilized on concanavalin A–coated magnetic beads and incubated in antibody buffer [wash buffer supplemented with 0.1% Triton X-100, 2 mM EDTA, and 100 μg mL⁻¹ BSA (NEB)] containing the appropriate primary antibody for 2 h at room temperature, followed by incubation with secondary antibody for 1 h at room temperature. Cells were washed once with TX-wash buffer [wash buffer containing 0.1% Triton X-100] and once with 300-TX-wash buffer [TX-wash buffer containing 300 mM NaCl], and then incubated with CUTANA™ pAG-Tn5 (EpiCypher). Tagmentation was performed at 37 °C for 1 h in tagmentation buffer [300-TX-wash buffer supplemented with 10 mM MgCl2 and 5 ng mL⁻¹ CUTANA™ E. coli spike-in DNA (EpiCypher)]. DNA was released in 2x release buffer [20 mM HEPES-KOH (pH 7.5), 20 mM EDTA, 2% SDS, and 2.5 μg mL⁻¹ proteinase K (Roche)] and incubated at 55 °C for 2 h. DNA was purified using a QIAquick PCR purification kit (QIAGEN), amplified with indexed primers, size-selected using SPRIselect beads (Beckman Coulter).
GEO
GSM9834067_r1