title: GSM9057750: A549, RPSA_KO, IAV_12h, CUT&Tag, rep 2; Homo sapiens; OTHER
library_name: GSM9057750
library_construction_protocol: Cells were harvested, counted and centrifuged for 3 min at 600g at room temperature. Aliquots of cells (60–500,000 cells), were washed twice in 1.5mL Wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5mM Spermidine; 1× Protease inhibitor cocktail) by gentle pipetting. Concanavalin A coated magnetic beads (Bangs Laboratories) were prepared and 10 µL of activated beads were added per sample and incubated at RT for 15min. We observed that binding cells to beads at this step increases binding efficiency. The unbound supernatant was removed. To amplify libraries, 21 µL DNA was mixed with 2 µL of a universal i5 and i7 primer, 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 5min (gap filling); 98 °C for 30 s; 14 cycles of 98 °C for 10 s and 63 °C for 30s; final extension at 72°C for 5 min and hold at 4 °C. Post-PCR clean-up was performed by adding 0.9 × volume of Ampure XP beads (Beckman Counter), and libraries were incubated with beads for 15 min at RT, washed twicegently in 80% ethanol, and eluted in 20 µL ddH2O.