Cut&Tag of Human BRD4 in HeLa cells
We performed the Cut&Tag experiments according to the manufacturers instructions (Vazyme, cat. no. TD901-TD902). HeLa Cells were harvested, counted and centrifuged for 3 min at 600g at RT. Aliquots of cells (about 100000 cells) were washed twice in 1.5 mL Wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1 Protease inhibitor cocktail) by gentle pipetting. 10 L of Concanavalin A beads (Bangs Laboratories) were activated, added per sample, rotated, and incubated at RT for 15 min. The unbound supernatant was removed and bead-bound cells were resuspended in 50 L Dig-wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1 Protease inhibitor cocktail; 0.05% Digitonin) containing 2 mM EDTA and a 1:50 dilution of the appropriate primary antibody. Primary antibody incubation was performed on a rotating platform for 2 h at RT. The primary antibody was removed by placing the tube on the magnet stand to clear and pulling off all of the liquid. Second antibody (goat anti-Rabbit IgG) was diluted 1:50 in 50 L of Dig-Wash buffer and incubated at RT for 30 min. Cells were washed 3 for 5 min in 800 L Dig-Wash buffer to remove unbound antibodies. A 1:200 of pG-Tn5 adapter complex (~0.04 M) was prepared in Dig-300 Buffer (0.05% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1Protease inhibitor cocktail) and added to the cells with gentle vortexing. Then the cells were rotated and incubated with transposase at RT for 1 h. Cells were washed 3 for 5 min in 800 L Dig-300 Buffer to remove unbound pG-Tn5 protein. Next, cells were resuspended in 100 L Tagmentation buffer (10mM MgCl2 in Dig-300 Buffer) and incubated at 37 C for 1 h. 10 L of 0.5 M EDTA, 3 L of 10% SDS and 2.5 L of 20 mg/mL Proteinase K was added to the sample, which was incubated at 55 C for 60 min to stop tagmentation. Then the total DNA was extracted using Phenol-chloroform-isoamyl alcohol 25:24:1 (Invitrogen, cat. no. 15593049). To amplify libraries, 21 L DNA was mixed with 2 L of a uniquely barcoded i5 and a i7 primer, (Vazyme, cat. no. TD202) using a different barcode for each sample. A volume of 25 L NEBNext HiFi 2 PCR Master mix was added and mixed. The sample was placed in a Thermocycler with a heated lid using the following cycling conditions: 72 C for 5 min (gap filling); 98 C for 30 s; 14 cycles of 98 C for 10 s and 63 C for 30 s; final extension at 72 C for 1 min and hold at 4 C. Post-PCR clean-up was performed by adding 1.1 volume of Ampure XP beads (Beckman Counter), and libraries were incubated with beads for 10 min at RT, washed twice gently in 80% ethanol, and eluted in 23 L 10mM Tris pH 8.0. Measure the concentration of each sample using Invitrogen Qubit 4 and Quantify the library yield. Determine the size distribution of libraries by Fragment Analysis capillary electrophoresis, using 2 L following the manufacturers instructions. Run a bead selection to choose the DNA fragment size from 300 to 500 bp using Ampure XP beads. Then the library was pooled together with equal molar and perform paired-end sequencing. The paired-end sequencing was performed on a single lane of an Illumina NovaSeq system yielding 150-bp paired-end (PE150) reads.