GSM9262129: rat tenocyte H4K8_LA; Rattus norvegicus; OTHER
100000 cells were washed twice gently with Wash Buffer. 10μL Concanavalin A coated magnetic beads were added per sample and incubated for 10min. Remove unbound supernatant and resuspended bead bound cells with Primary Antibody Buffer and a 1:50 dilution of primary antibody (PTM-1419RM, PTM-1415RM, PTM-1417RM) or IgG control antibody (rabbit IgG, CST, 2729S) incubated overnight at 4℃. The primary antibody was removed using magnet stand. Sencondary antibody (Goat anti-rabbit IgG H&L pAb: PTM-6252) was diluted 1:200 in Antibody Buffer and cells were incubated at RT for 60 min. Cells were washed using the magnet stand 2-3 times in Antibody Buffer. A 1:80 dilution of pA-Tn5 adapter complex was prepared in ChiTag Buffer and incubated with cells at RT for 1h. Cells were washed 2–3× for 5 min in ChiTag Buffer. Then cells were resuspended in Tagmentation Buffer and incubated at 37 °C for 1 h. DNA was purified using Tagment DNA Extract Beads. To amplify libraries, 20μL DNA was mixed with 2.5μL of a universal i5 and a uniquely barcoded i7 primer. A volume of 25 μL 2×HiFi AmpliMix was added and mixed. The sample was placed in a Thermocycler with a heated lid using the following cycling conditions: 72 °C for 3 min; 98 °C for 30 s; 13 cycles of 98 °C for 15 s, 63 °C for 8 s, 72 °C for 5 s; final extension at 72 °C for 2 min and hold at 10 °C. Library clean-up was performed by NovoNGS DNA Clean Beads. CUT&Tag was performed using the NovoNGS CUT&Tag 4.0 High-Sensitivity Kit (for Illumina) (Cat. No.: N259-YH01) following the manufacturer's protocol.