ATAC-seq of iPSC-derived Motor Neurons
ATAC-seq was performed as described in Dong et. al 2020. 100,000-200,000 cells. Cells were resuspended and centrifuged at 500 RCF for 10 minutes at 4?°C. The supernatant was aspirated.The cells were resuspended in 300 µl DNaseI solution (20?mM Tris pH 7.4, 150?mM NaCl, 1× reaction buffer with MgCl2, 0.1U/µl DNaseI) on ice for 5 minutes. Subsequently, 1 ml of PBS (0.04% BSA) was added, and the mixture was centrifuged at 500 rcf for 10 minutes at 4?°C. The cells were washed twice in PBS+BSA and resuspended in 100 µl ATAC-seq RSB (10?mM Tris pH 7.4, 10?mM NaCl, 3?mM MgCl2 in water) with 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin and incubated on ice for 3 minutes to lyse. After lysis, 1 mL of ATAC-seq RSB with 0.1% Tween-20 was added and mixed by inversion. Next, the nuclei were collected by centrifugation at 1000 RCF for 10?min at 4?°C. The nuclei were resuspended in 20?µL 2× TD buffer (20?mM Tris pH 7.6, 10?mM MgCl2, 20% Dimethyl Formamide), and 50,000 nuclei were transferred to a tube with 2× TD buffer filled up to 25?µL. 25?µL of transposition mix [2.5?µL Transposase (100?nM final) (Illumina, 20034197), 16.5?µL PBS, 0.5?µL 1% digitonin, 0.5?µL 10% Tween-20, and 5?µL water] was added. The transposition reactions were mixed and incubated for 30 minutes at 37?°C with gentle tapping every 10?min. Reactions were purified with the Zymo DNA Clean and Concentrator-5 kit (Zymo Research, D4014). The ATAC-seq library was amplified for nine cycles with Nextara primers in a PCR machine. The PCR reaction was purified with Sera-Mag Select beads (Cytiva, 29343057) using double size selection with 27.5?µL beads (0.55× sample volume) and 50?µL beads (1.55× sample volume). The ATAC-seq libraries were quantitated by Qubit assays and sequenced by an Illumina NextSeq 500 platform.