title: Disruption of ADNP-KDM1A-GTF2I complex drives neural differentiation imbalance in Helsmoortel-Van der Aa syndrome [multi-omic]
design: Disruption of ADNP-KDM1A-GTF2I complex drives neural differentiation imbalance in Helsmoortel-Van der Aa syndrome [multi-omic]
library_construction_protocol: Cortical organoids were dissociated using Dissociation Buffer composed of 0.5% BSA, 2 mM EDTA, 0.1% Accutase, 0.4 mg/ml Collagenase/Dispase (Sigma-Aldrich, 10269638001), 0.05 mg/ml DNAse I (Sigma-Aldrich, 10104159001) diluted into final volume of PBS, and filtered with 0.22 μm filters. Few homogeneous-sized organoids were incubated for approximately 40 minutes on a rotating wheel at 37°C with 1 ml of Dissociation Buffer, manually pipetting every 10 minutes. Organoids were transferred to a new Eppendorf leaving behind undissociated pieces, and then centrifuged at 300g for 5 minutes at 4°C. Cells were resuspended in 1 ml of PBS-BSA 0.04% and filtered with 40 μm Flowmi cell strainer (Bel-Art, H13680-0040) to remove the majority of debris. Cells were counted manually and then multiplexed together with other samples to obtain a final number of 1 million cells, with equal representation from each sample (e.g., 250K cells in case of 4 samples multiplexed).  Cortical organoids were generated using an adaptation of the previously described protocol published by Trujillo et al.  Multiplexed cell solution was washed twice with PBS-BSA 0.04% and pellet was resuspended in 100 μL of Lysis Buffer [10mM Tris-HCl (pH 7.4), 10mM NaCl, 3mM MgCl2, 0.1% Tween-20 (Bio-Rad, 1662404), 0.1% Nonidet P40 Substitute (Sigma-Aldrich, 74385), 0.01% Digitonin (Thermo Fisher, BN2006), 1% BSA, 1 mM DTT (Sigma-Aldrich, 646563), 1U/μL RNase inhibitor (Sigma-Aldrich, 3335399001), nuclease-free water], and incubated 5 minutes on ice. Lysis Buffer was washed away three times with 1 ml of Wash Buffer [10mM Tris-HCl (pH 7.4), 10mM NaCl, 3mM MgCl2, 1% BSA, 0.1% Tween-20, 1 mM DTT, 1U/μL RNase inhibitor, nuclease-free water]. Assuming 50% nuclei loss during lysis, nuclei were resuspended in Diluted Nuclei Buffer [1X 10x Genomics Nuclei Buffer, 1 mM DTT, U/μL RNase inhibitor, nuclease-free water] according to the reference table provided by 10X protocol appendix.  The volume of Nuclei Diluted Buffer is critical to fit the right range of concentration based on the number of targeted nuclei recovery, therefore avoiding overcrowding of the Chromium machine during tagmentation and GEM preparation steps. Resuspended nuclei were passed again through a Flowmi cell strainer and counted to check for the right concentration feasible for the number of targeted nuclei. 5,000 was the number of targeted nuclei recovery for each multiplexed sample in all the experiments (e.g., 20,000 nuclei were targeted in the reactions with 4 multiplexed samples).