GSM6502570: MM057_BORIS_no_dox_Rep1; Homo sapiens; ATAC-seq
Cells were detached from the culture dish with citric saline (270mM KCl, 30mM sodium citrate), passed through a 40um filter and counted using a hemocytometer to collect 50.000 cells for all four experimental conditions. To isolate nuclei, cells were first lysed for 20 min on ice in lysis buffer 1 (0.1% Sodium citrate tribasic dehydrate (Sigma, S1804) and 0.1% Triton X-100 (Bioshop, TRX777.100)) followed by 20 min lysis on ice in lysis buffer 2 (10mM Tris-HCl pH7.4, 10mM NaCl, 3mM MgCl, 0.1% Igepal CA630 (Bioshop, NON999.500) and 0.1% Tween-20 (Bioshop, TWN510.500). The nuclei were pelleted by centrifugation for 10min at 600g (4°C) and supernatant was carefully removed to reduce mitochondrial DNA (mtDNA) content. For the transposase reaction, the nuclei pellet was re-suspended in 25μl transposase reaction mix containing of 4μl Tn5 transposase in 1x transposase reaction buffer (Nextera DNA library kit, Illumina, FC-121–1030) and 0.1% Tween-20 (Bioshop, TWN510.500), and incubated for 30min at 37°C with gentle shaking (400rpm). Immediately following transposition, DNA was purified using the MinElute PCR purification Kit (Qiagen, 28004), according to manufacturer's instructions. Library fragments were amplified in two steps. First, 5 cycles of PCR were performed using NEBNext® High-Fidelity PCR master mix (New England BioLabs, M0541) with 1.25μM barcoded Nextera PCR primers (Supp 10.). in a final volume of 50μl on a BioRad C1000 Thermal Cycler. The cycling program used was as follows: 5 min at 72°C, 30 sec at 98°C, 5 times cycling for 10sec at 98°C, 30sec at 63°C and 1 min at 72°C. To reduce GC and size bias during library amplification, 5μl from the PCR mix was used for a 15μl qPCR side-reaction containing the same reagents with the addition of SYBRgreen (9X, Life Technologies, S7563) to determine the number of additional PCR cycles and stop amplification prior to saturation. The CFX96 TouchTM (Bio-Rad, 1855196) qPCR machine was used with the following cycling conditions: 30 sec at 98°C, 20 times cycling for 10sec at 98°C, 30sec at 63°C and 1 min at 72°C followed by melt curve analysis. Using the CFX ManagerTM Software (Bio-Rad), the fluorescent intensity was plotted versus cycle number and the additional number of cycles was established as the cycle number corresponding to 1/4th of the maximum fluorescent intensity. Next, the initial PCR reaction mix was amplified for 3 additional PCR cycles. Library preparation for each experimental condition was performed in triplicate and samples were combined post PCR amplification for quality controls and sequencing. the library was size selected using KAPA Pure Beads (0.55X-1.8X; Roche, 7983298001) to enrich for 150-750 bp fragments. DNA of the libraries was submitted to the McGill University and Genome Québec Innovation Centre and fragment distribution was assessed by TapeStation (Agilent), libraries were quantified using Quant-iTTM PicoGreen® dsDNA Reagent® (ThermoFisher Scientific), pooled and sequenced paired-end (100bp read length) on one lane of an Illumina HiSeq4000.