title: Illumina HiSeq 4000 paired end sequencing; Open chromatin profiling in the U2OS cell
design: Open chromatin profiling in the U2OS cell
library_construction_protocol: Cells were lysed 48 h after transfection in cold lysis buffer (10mM Tris-HCL, pH 7.4, 10mMNaCl, 3mM MgCl2 and 0.1% Igepal). DNA was purified using Qiagen mini elute kit and using Ampure XP beads following the manufacturer's instructions. Nuclei was concentrated by centrifugation at 500 g for 10 minutes at 4°C. The nuclei pellet was resuspended in nuclease free water, diluted 1:10 and quantified. The equivalent volume for 50,000 nuclei was made up to 22.5 ul with nuclease free water. Nuclei were incubated for 30 minutes at 37°C, 300 rpm, with 25 ul of 2x TD buffer and 2.5 ul tagmentation ezyme (Illumina transposase FC-121-1030). Library fragments were amplified using standard PCR and Nextera primers (Illumina) by adding 25 ul of 2x NEBnext master mix, 2.5 ul forward primer, 2.5 ul reverse primer and 20 ul transposased DNA.  PCR was run for 5 cycles, 72°C, 5 minutes; 98°C 30 seconds; cycle of 98°C 10 seconds, 63°C 30 seconds, 72°C 1 minute, paused at 4°C. To determine the total number of cycles needed for the amplified library, 5 ul of the PCR mix were taken and added to a new mix containing 5 ul 2X master mix, 1 ul of each primer, 0.6 ul of 10X SYBR green and 2.4 ul H2O. In a qPCR machine, 20 cycles were run, the total number of cycles for the ATAC library was chosen according to qPCR cycle that gave 1/3 of saturation.