GSM8048449: CnR_NTC_2; Homo sapiens; OTHER
DNA binding sites of RUNX1 were determined by using the CUT&RUN method, originally established by Skene and Henikoff in 201749, as described in the EpiCypher CUT&RUN Protocol v2.0 and in Boos et al. 35 with modifications for cell type and antibody. Here, 500,000 NTC, SMANTIS KO, and RUNX1 KO THP-1 were used and first washed with wash buffer (20 mM HEPES pH 7.9, 150 mM NaCl, 500 nM spermidine 1X Roche Protein Inhibitor Cocktail) at RT. Cells were then resuspended in wash buffer and immobilized on magnetic beads. Therefore, cells were incubated with 10 µl BioMag®Plus Concanavalin A (ConA) beads (Polysciences, 86057-3) for 10 min at RT. Beads were washed once on a magnetic rack in 100 µl antibody buffer (wash buffer, 0.25% Digitonin, and 2 mM EDTA) and 1 µl RUNX1 antibody (ab240639, Abcam). After an over-night incubation of the beads with antibody with gentle shaking at 4°C, the beads were washed twice with 200 µl 0.25% Digitonin wash buffer followed by resuspension and incubation in Digitonin wash buffer containing 2 µl CUTANA™ pAG-MNase (15-1016, EpiCypher) on ice for 30 min. After washing the samples twice, the samples were resuspended in 100 µl Digitonin wash buffer with addition of 2 µl CalCl2 (final concentration of 100 mM) and incubated for 2 h at 4°C with gentle shaking. To stop the enzymatic reaction, samples were incubated in 33 µl stop buffer (340 mM NaCL, 20 mM EDTA, 4 mM EGTA, 0.25 % Digitonin, 100 µg/mL RNase A, 50 µg/mL Glycoblue) for 10 min at 37°C. Samples were then placed on a magnetic rack and supernatant containing the CUT&RUN-enriched DNA was collected in a fresh 1.5 mL tube. DNA purification was performed by using 5X volume of binding buffer (20 mM HEPES pH 7.9, 20 mM KCL, 1 mM CaCl2, 1 mM MnCl2). The pH was adjusted with sodium acetate before transferring the sample into a purification column (58002, ActiveMotif). Samples were then centrifuged at 11,000 xg for 30 sec. The column was washed with 750 µl wash buffer and dried by centrifugation for 2 min. The DNA was eluted in 25 µl elution buffer and concentration was measured with Qubit 3.0 Fluormeter (Life Technologies). For library prepration, DNA fragments were cleaned up by 1xSPRI bead cleanup and used as input for SMARTer ThruPLEX DNA-seq Kit (Takara Bio) following the manufacturer protocol with exception of individual library cleanup after final PCR. Libraries were mixed in equimolar ratios and sequenced on NextSeq2000 platform using P3 flowcell.