GSM4655323: ATAC_XCR_GFPneg_Day9; Mus musculus; ATAC-seq
For the isolation of Day 0 time point of ATAC-seq analysis, 20,000 cells were sorted into a 1,5 ml centrifuge tube, followed by initial steps of Omni-ATAC-seq protocol. Remaining cells were plated for reprogramming directly after sorting, 50.000 cells per one well of a 12 well plate. Reprogramming was induced by doxycycline (2 μg/ml final) in mouse ESC medium in the presence of ascorbic acid (50 μg/ml final). The medium together with doxycycline and ascorbic acid was replaced every two days and maintained throughout entire reprogramming experiments. To derive iPSCs from picked colonies, the same conditions were used with dox and AA withdrawal after picking. Assay for transposase accessible chromatin (ATAC) followed by sequencing was performed using the Omni-ATAC protocol (Corces et al., 2017). Briefly, 20,000 sorted Day 0 (Xi-GFP),  FUT4+/Xi-GFP Day 8, 9, 10, 12 reprogramming intermediates, and iPSC were pelleted at 500 RCF at 4°C for 5 min in a fixed-angle centrifuge, and then the cells were gently washed once with 50 µl of cold PBS. Next, the cell pellets were resuspended in 50 µl of ATAC-lysis buffer (10mM Tris HCl pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20, 0.1% NP40, and 0.01% Digitonin) and incubated on ice for 3 min. Wash out lysis with 1 ml of cold ATAC-lysis buffer containing 0.1% Tween-20 but No NP40 or digitonin and invert tube 3 times to mix. Nuclei were pelleted at 500 RCF for 10 min at 4°C in a fixed-angle centrifuge. After discarding all supernatant, nuclei were resuspended in 50 µl of transposition mixture (25 ul 2x TD buffer, 2.5 ul transposase (100 nM final), 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, and 5 ul H2O) (Nextera DNA Sample Preparation Kit, Illumina, FC-121-1030). The reaction was performed at 37°C for 30 minutes in a thermomixer with 1000 RPM mixing. The transposed DNA was purified using a Zymo DNA Clean and Concentrator-5 Kit (D4014). DNA libraries were PCR amplified using NEBNext HighFidelity 2x PCR Master Mix (Bioke, M0541), and size selected for 200 to 800 bp using homemade Serapure beads (Rohland and Reich, 2012). Library concentrations were quantified with the KAPA Library Quantification Kit (KK4854), and equimolar amounts were pooled for paired-end sequencing on an Illumina NextSeq 500 instrument (Illumina) to yield ~100 million of 75 bp long reads per sample.