title: Sample 12
organism: Homo sapiens
description: Protocols: FUCCI hESCs were washed with PBS and collected by incubating with Cell Dissociation Buffer (Gibco) for 10 min at 37°C. Cells were washed with 1% PBS/BSA and incubated in 1% PBS/BSA with anti-TRA-1-60-antibody mouse monoclonal IgM (1:100, Santa Cruz Biotechnology, Inc.) for 20 min on ice, together with the secondary antibody, Alexa Fluor® 647 goat anti-mouse IgM (1:1,000, Thermo Fisher) to remove background differentiation. Cells were washed with PBS, re-suspended in 3ml of PBS and sorted into the different cell cycle phases using BD FACS AriaTM III, BD Fusion or BD Influx sorters by the Cambridge NIHR BRC Cell Phenotyping Hub. Samples were generated from two independent experiments, 100,000 cells per sample, as previously described in (Kumasaka et al., 2016) with some modifications. In brief, cells were washed with ice-cold PBS and incubated with 500μl of ice-cold sucrose buffer (10mM Tris-Cl pH 7.5, 3mM CaCl2, 2mM MgCl2 and 0.32M sucrose) for 12 min on ice. To collect the nuclei, Triton X-100 was added to a final concentration of 0.5% and cells incubated for additional 6 min on ice after mixing. Samples were then resuspended in 1ml of sucrose buffer and transferred to a fresh 1.5 ml Eppendorf tube. The nuclei were centrifuged at 1500g for 3 min at 4°C and the supernatant discarded. Nuclei pellets resuspended in 50μl of Nextera DNA Sample Preparation Kit (Illumina FC-121-1030) for tagmentation, comprising 25μl 2x Tagment DNA buffer, 20μl nuclease-free water and 5μl Tagment DNA Enzyme 1. The tagmentation reaction mixture was transferred in a 1.5ml low-binding tube and incubated at 37°C for 30 min and stopped by the addition of 250μl Buffer PB (Qiagen). The tagmented chromatin was purified using the MinElute PCR purification kit (Qiagen 28004), according to the manufacturer's instructions, eluting in 11.5μl of buffer EB (Qiagen). For PCR amplification, 10μl of the tagmented chromatin was mixed with 2.5μl of Nextera PCR primer cocktail and 7.5μl of Nextera PCR mastermix (Illumina FC-121-1030) in low-binding PCR tube. Primers were from the Nextera Index kit (Illumina FC-121-1011), using 2.5μl of an i5 primer and 2.5μl of an i7 primer per PCR, totalling 25μl. PCR amplification was performed as follow: 72°C for 3 min and 98°C for 30 sec, followed by 12 cycles at 98°C for 10 sec 63°C for 30 sec and 72°C for 3 min. The sample volumes were then increased to 50μl by adding Qiagen EB buffer. The PCR primers were removed with 1x 0.9:1 SPRI beads (Beckman Coulter, Cat no. A63880) according to manufacturer's instruction and samples eluted in 20μl. Following amplification, excess of primers was removed running the samples of 50ml 1% agarose TAE gel at 90V for 25 min, and size selection obtained by cutting from 120bp to 1kb. MinElute Gel Extraction kit (Qiagen 28604) was used to extract DNA, eluted in 20μl of buffer EB. Library preparation and sequencing were performed at the Wellcome Sanger Institute next-generation sequencing facility. 13 ATAC-seq libraries were prepared with one of i5 and i7 Nextera tags combination, and pooled equimolarly.