GSM5121218: KLF4_naive_rep1; Homo sapiens; OTHER
200,000 cells per sample replicate were washed in Wash Buffer [1 mL 1 M HEPES pH 7.5 (Sigma Aldrich, H3375), 1.5 mL 5 M NaCl (Sigma Aldrich, S5150), 12.5 μL 2 M Spermidine (Sigma Aldrich, S2501), 1 Roche Complete Protease Inhibitor EDTA-Free tablet (Sigma Aldrich, 5056489001), and bring the final volume to 50 mL with dH2O], then immobilized on 10 ul of Concanavalin A-coated beads (Bangs Laboratories). Cells were cleared on a magnetic rack, then permeabilized with Dig-wash buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine and 1x protease inhibitor cocktail containing 0.05 % Digitonin]. The cells were then incubated with primary antibody KLF4 (R&D, AF3640, 1:100) at 4°C overnight. The primary antibody was cleared on a magnetic rack. Rabbit anti-Goat IgG secondary antibody was diluted 1:100 in Dig-wash buffer and incubated at RT for an hour. Cells were cleared on a magnetic rack and washed with 1 mL of Dig-wash buffer. A 1:200 diluted of pA-Tn5 adapter complex was prepared in Dig-300 Buffer [20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM Spermidine and 1x protease inhibitor cocktail containing 0.05 % Digitonin]. Cells were cleared on a magnetic rack and incubated by adding 100 ul of pA-Tn5 at RT for 1 hour. Cells were washed with 1 mL of Dig-300 buffer, resuspended in 300 ul of Tagmentation buffer [10 mM MgCl2 in Dig-300 Buffer], and incubated at 37℃ for 1 hour. 10 ul of 0.5 M EDTA, 3 ul of 10% SDS and 2.5 ul of 20 mg/mL Proteinase K was added to each reaction to stop the tagmentation at 55℃ for an hour. DNA was purified using phenol/chloroform/isoamyl alcohol (PCI) extraction followed by chloroform extraction and precipitated with glycogen and ethanol. DNA was pelleted with a high-speed spin at 4℃, washed, air dried for 5 min and resuspended in 50 ul of double-distilled water (Kaya-Okur et al., 2019). The DNA was PCR amplified using i5 and i7 indexing primers, and cleaned up with AMPure XP beads (Beckman Coulter, A63880). Agilent 4200 TapeStation analysis was performed for size distribution of libraries and as desired aiming for a final concentration, libraries were mixed to achieve equal representation. Paired-end Illumina sequencing on the barcoded libraries was used by following the manufacturer's instruction.