Instrument: HiSeq X Ten
Strategy: ATAC-seq
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: A total of 50,000 cells (reprogrammed with O4SK, O6SK and O4defS2SK retroviruses) were collected at days 1 and 5 of reprogramming in replicates. Cells were washed once with 50 μL of cold PBS; centrifuged at 500 x g for 5 min at 4°C and cell pellets were resuspended in 50 μL cold ATAC lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630) by slowly pipetting up and down and immediately spun down at 500 x g for 10 min at 4°C to collect nuclei. Nuclei were washed in 1x PBS and subsequently re-suspended in 50 μL transposition reaction mix (25 μL 2 x TD reaction buffer, 22.5 μL nuclease-free water, 2.5 μL Tn5 transposase) of Nextera DNA library preparation kit (FC-121-1030, Illumina). Samples were incubated at 37°C for 30 min and DNA was isolated using minElute Kit (Qiagen). The transposed DNA was then amplified with custom primers as described for 1 cycle of 72°C for 5 min, 98°C for 30 sec followed by 5 cycles of 98°C for 10 sec, 63°C for 30sec, 72°C for 1 min. To determine the suitable number of cycles required for next round of PCR the library was assessed by quantitative PCR. Libraries qualities were assessed using Bioanalyzer high sensivity DNA analysis kit (Agilent) followed by paired-end sequencing with the length of 150 nucleotides using Hiseq X10 (illumina) at Annoroad Gene Technology (http://en.annoroad.com/). The libraries were generated using the Nextera DNA Library Prep kit (Illumina)