title: GSM8504288: ES,  0h, rep1; Mus musculus; ATAC-seq
library_construction_protocol: Reprogramming XEN cells were collected at Day 7, Day 14, Day 28 of reprogramming using fluorescence-activated cell sorting (FACS) to enrich for cells undergoing lineage conversion based on key lineage marker expression. Starting XEN and fully reprogrammed XEN-iPS cells were also assayed. ES cells converting to iXEN cells were also collected at 9h, 24h and 48h of reprogramming. A similar FACS enrichment strategy was used for the 9h timepoint as was used for reprogramming XEN cells. Starting ES and fully converted iXEN cells were additionally sampled. All reprogramming XEN cells were dissociated using 0.25% Trypsin-EDTA, and other cells using 0.05% Trypsin-EDTA (i.e. starting and end XEN, ES, iPS and iXEN populations, and converting ES-to-iXEN cells). Cells taken for sorting were filtered through a 0.35um nylon mesh strainer to achieve single cell suspensions. Cells were centrifuged at 400g for 5 mins at room temperature and washed twice with wash buffer (5% FBS in PBS without Ca2++ and Mg2++). Cell pellets were then resuspended in 100 µl staining buffer (wash buffer with diluted antibodies) and incubated at room temperature for 30 mins protected from light. Following incubation, cells were washed twice with wash buffer. Cell pellets were finally resuspended in wash buffer containing 1µg/ml 4′,6-diamidino-2-phenylindole (DAPI). Finally, cells were resuspended at a final concentration of 5-10x106/ml in wash buffer and sorted using SORP FACSAria IIu (BD Biosciences) with a 100µm nozzle at 20psi. Dissociated or sorted cells were washed in PBS + 0.04% BSA and processed for nuclei isolation following manufacturer's instructions (Demonstrated Protocol: Nuclei Isolation for Single Cell ATAC Sequencing; Document CG000169 Rev D). Final nuclei preparations were loaded on a Chromium Controller targeting 10,000 nuclei, and processed in singlicate. Libraries were generated following the manufacturer's instructions (10x Genomics Chromium Next GEN Single Cell ATAC Reagent Kit Guide v1.1). Briefly, isolated nuclei were resuspended in diluted nuclei buffer and taken for the transposition reaction. Transposed nuclei were then diluted in master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The DNA fragments from the nuclei in every single GEM were PCR linear amplified and appended with an Illumina P5 primer sequence, a Read 1N sequence, and the 10x barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, and the appropriated size fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.