GSM9897042: hiPSC-derived liver organoid 46 n2  fasted; Homo sapiens; ATAC-seq
(1) hiPSCs were dissociated using an accutase solution (Sigma, #A6964) for 5 min at 37 °C, followed by the addition of 10 mg/mL collagenase IV (Sigma) in TrypLE dissociation reagent (Thermo Fisher) for a further 20 min. A total of 50,000 live cells were collected.                                                  (2) Liver organoids at day 20 were manually picked using a microscope (Vision Engineering) and transferred into an ultra-low-binding 6-well plate (BD Falcon) at a density of 200 organoids/well for 1 day before ATAC experiment. The day after, the cell supernatant was removed and kept for further analysis, and organoids were processed for single-cell dissociation. Liver organoids  were dissociated using enzymatic digestion with Ca2+Mg2+-free phosphate buffer and dissociated for 5 min at 37 °C using first 2 mL TrypLE dissociation reagent animal-free (Thermo Fisher) followed by the addition of 10 mg/mL collagenase IV (Sigma) solution for 20 min. When complete, the dissociation was blocked using Ca2+Mg2+-containing HBSS buffer supplemented with 1% BSA (Thermo Fisher) and the cell suspension was passed through a 37 µm strainer to remove possible cell aggregates. 50 000 single living cells were collected and centrifuged for 10 min at 500 G at 4 °C. The pelleted cells were resuspended in a transposase reaction mix containing digitonin (25 µL of 2x TD buffer, 5 µL of TDE1 (Illumina), 1 µL of 0.5% digitonin (Promega) and 19 µL of nuclease-free water). The transposition reaction was then carried out for 30 min at 37 °C. The samples were then purified immediately using a DNA Clean and Concentrator-5 kit (Zymo Research). Following purification, each sample was dual indexed using i7 and i5 primers from the Nextera Index Kit (Illumina), then amplified using the Nextera DNA Library Prep Kit (Illumina) under the following PCR conditions: 72 °C for 3 min, 98 °C for 30 sec, followed by 11 cycles of 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 3 min. Libraries were double size-selected using Beckman Coulter™ Agencourt AMPure XP beads at 0.5x and 1.3x ratios. The quality of the libraries was assessed using a TapeStation, and the libraries were quantified using a NEBNext® Library Quant Kit for Illumina® (New England Biolabs). Sequencing was performed on an Illumina NovaSeq 6000 platform in 2 × 51 paired-end mode. Trimming and demultiplexing were performed using Illumina bcl2fastq2.