GSM5745744: CYCT1-NHS-Rep1; Homo sapiens; OTHER
We performed the Cut&Tag experiments according to the manufacturer's instructions (Vazyme, cat. no. TD901-TD902). HeLa Cells were harvested, counted, and centrifuged for 3 mins 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 μL of Concanavalin A beads (Bangs Laboratories) were activated and added to each sample. The mixture was rotated and incubated for 15 mins at RT. The supernatant was removed and bead-bound cells were resuspended in 50 μL 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 primary antibodies. The incubation was rotated for 2 h at RT. Then primary antibodies were removed by pulling off all of the supernatants after placing the tube on the magnet stand. The second antibody (goat anti-Rabbit IgG) was diluted 1:50 in 50 μL of Dig-Wash buffer and incubated at RT for 30 mins. Cells were washed 3× for 5 mins in 800 μL Dig-Wash buffer to remove unbound antibodies. A 1:200 pG-Tn5 adapter complex (~0.04 μM) was prepared in Dig-300 Buffer (0.05% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1×Protease inhibitor cocktail) and added to cells. Then cells were rotated and incubated with transposase at RT for 1 h. Cells were washed 3× for 5 min in 800 μL Dig-300 Buffer to remove unbound pG-Tn5 protein. Next, cells were resuspended in 100 μL Tagmentation buffer (10 mM MgCl2 in Dig-300 Buffer) and incubated at 37 °C for 1 h. 10 μL of 0.5 M EDTA, 3 μL of 10% SDS and 2.5 μL 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 μL DNA was mixed with 2 μL of a uniquely barcoded i5 and an i7 primer, (Vazyme, cat. no. TD202) using a different barcode for each sample. A volume of 25 μL 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 mins at RT, washed twice gently in 80% ethanol, and eluted in 23 μL 10mM Tris pH 8.0. The concentration of each sample was measured using Invitrogen Qubit 4. The size distribution of libraries was determined by fragment analysis capillary electrophoresis, using 2 μL sample following the manufacturer's 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 an equal molar and performed paired-end sequencing on a single lane of an Illumina NovaSeq system yielding 150-bp paired-end (PE150) reads.