Single-nucleus RNA sequencing of BIN1 WT, HET and KO hiPSC-derived neural cells

Count tables showing the number of counts per nucleus in 190 days-old BIN1 WT, HET and KO human cerebral organoids (org_counts_processed) and in BIN1 WT and KO human induced neural cells grown in 2D (spontaneous differentiation or ASCL1-induction). Nuclei isolation and Hash-tagging with oligonucleotides steps were realized on ice with pre-cold buffers and centrifugations at 4°C. 6.5-month-old BIN1 WT, HET, and KO organoids were processed as previously (Lambert et al., 2022). 4-week-old cultured ASCL1-induced or 6-week-old spontaneously differentiated BIN1 WT and KO 2D neural cell cultures were washed in the imaging plate wells with 500 µL of Deionized Phosphate Buffer Saline 1X (DPBS, GIBCO™, Fisher Scientific 11590476). Cells were resuspended with wide bore tips in 500 μL Lysis Buffer (Tris-HCL 10mM, NaCl 10mM, MgCl2 3mM, Tween-20 0,1%, Nonidet P40 Substitute 0,1%, Digitonin 0,01%, BSA 1%, Invitrogen™ RNAseout™ recombinant ribonuclease inhibitor 0,04 U/μL). Multiple mechanical resuspensions in this buffer were performed for a total lysis time of 15 mins., 500 μL of washing buffer was added (Tris-HCL 10mM, NaCl 10 mM, MgCl2 3 mM, Tween-20 0.1%, BSA 1%, Invitrogen™ RNAseout™ recombinant ribonuclease inhibitor 0,04 U/μL) and the lysis suspension was centrifuged 8 mins. at 500 g (used for all following centrifugation steps). Nuclei pellets were washed tree times with one filtration step by MACS pre-separation filter 20μm (Miltenyi Biotec).Nuclei pellets were resuspended in 100 μL of staining buffer (DPBS BSA 2%, Tween-20 0.01%), 10 μL of Fc blocking reagent HumanTruStainFc™ (422302, Biolegend) and incubated 5 min at 4°C. 1μl of antibody was added (Total-Seq™-A0453 anti-Vertebrate Nuclear Hashtag 3 MAb414 for the WT and Total-Seq™-A0454 anti-Vertebrate Nuclear Hashtag 4 MAb414 for the KO, 97286 and 97287 respectively, Biolegend) and incubated 15 mins. at 4°C. Nuclei pellets were washed three times in staining buffer with one filtration step by MACS pre-separation filter 20 μm (Miltenyi Biotec) to a final resuspension in 300 μL of staining buffer for Malassez cell counting with Trypan blue counterstaining (Trypan Blue solution, 11538886, Fisherscientific). Isolated nuclei were loaded on a Chromium 10X genomics controller following the manufacturer protocol using the chromium single-cell v3 chemistry and single indexing and the adapted protocol by Biolegend for the HTO library preparation. The resulting libraries were pooled at equimolar proportions with a 9 for 1 ratio for Gene expression library and HTO library respectively. Finally, the pool was sequenced using 100pb paired-end reads on NOVAseq 6000 system following the manufacturer recommendations (Illumina).