title: GSM8208831: 550_S5_PAX4 [ATAC-Seq]; Homo sapiens; ATAC-seq
library_construction_protocol: For flow cytometry, differentiating aggregates were incubated with Accutase for 5 minutes in a water bath and dispersed to single cells.  Cells were then stained with SYTOX-Blue for 5 min at RT. Cells were sorted using a FACS Aria III (Becton Dickinson) operated with BD FACSDiva software. For nuclei isolation and library construction, a low input nuclei isolation protocol adapted from 10X Genomics was performed. In brief, sorted cells were washed once with 1 mL PBS + 1% BSA, counted, centrifuged, and supernatant was aspirated. Subsequently, the washed cell pellet was resuspended in chilled lysis buffer with 0.1x detergent concentration (50 μL per sample) and placed on ice for 5 min. Then wash buffer (500 μL per sample) was added and nuclei were centrifuged. To gradually change from wash to diluted nuclei buffer, cells were washed once in a 1:1 mixture of wash buffer and diluted nuclei buffer and subsequently one with pure diluted nuclei buffer. The washed isolated nuclei were then resuspended in 7-10 μl diluted nuclei buffer and were directly added to the transposition reaction after quality control and counting. In all following steps, 10X Genomics' Single Cell Multiome ATAC and gene-expression protocols were followed according to the manufacturer's specifications and guidelines.