title: GSM8545760: HUVEC, BRG1 CUT&RUN, siJPX; Homo sapiens; OTHER
library_construction_protocol: 5X volume of binding buffer (20 mM HEPES pH 7.9, 20 mM KCl, 1 mM CaCl2, 1 mM MnCl2) was added to the samples and the pH adjusted with sodium acetate before being transferred to a purification column (ActiveMotif, 58002) and centrifuged at 11,000 xg for 30 sec. The column was then washed with 750 µl wash buffer and dried by centrifugation for 2 min. DNA was eluted with 25 µl elution buffer and the DNA concentration measured with a Qubit 3.0 Fluorometer (Life Technologies). Samples were brought to 50 µl with 0.1X TE buffer and DNA end preparation performed as instructed but with incubation at 20 °C for 20 min and then 58 °C for 45 min. Adaptor ligation was performed with a 1:10 dilution of adaptor (NEB, E6440S). For DNA purification, 0.9X Volume AMPure XP beads (Beckman Coulter, A63881) was added to the samples and incubated for 5 min at RT. Beads were washed twice with 200 µl 80 % ethanol and DNA eluted with 17 µl 0.1X TE buffer for 2 min at RT. PCR amplification of the eluted DNA was performed as described in the manufacturer's protocol but with the addition of 2.5 µl Evagreen (20X) for visualization of the amplification curves on an AriaMx Real-time PCR system (Agilent Aria 1.7). The denaturation and annealing/extension steps of the PCR amplification were performed for around 12 cycles and stopped before the curves plateaued. A cleanup of the PCR reaction was performed twice with 1.1X Ampure beads and eluted each time in 33 µl 0.1X TE buffer. DNA concentrations were measured with a Qubit (Thermo Fisher) and size distributions measured on a Bioanalyzer (Agilent).