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).
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Unique Molecular Index (UMI) Count Matrices for gene expression and for Hash Tag Oligonucleotide (HTO) libraries were generated using the CellRanger count (Feature Barcode) pipeline. Reads were aligned on the GRCh38-3.0.0 transcriptome reference (10x Genomics). Filtering for low quality cells according to the number of RNA, genes detected, and percentage of mitochondrial RNA was performed. For HTO sample, the HTO matrix was normalized using centered log-ratio (CLR) transformation and cells were assigned back to their sample of origin using HTODemux function of the Seurat R Package (v4). Then, normalizations of the gene expression matrix for cellular sequencing depth, mitochondrial percentage and cell cycle phases using the variance stabilizing transformation (vst) based Seurat:SCTransform function were performed. To integrate the datasets from independent experiments, the harmony R package (https://github.com/immunogenomics/harmony) was used. In order to integrate the datasets, the SCTransform normalized matrices was merged and PCA was performed using Seurat::RunPCA default parameter. The 50 principal components (dimensions) of the PCA were corrected for batch effect using harmony::RunHarmony function. Then, the 30 first batch corrected dimensions were used as input for graph-based cell clustering and visualization tool. Seurat::FindNeighbors using default parameters and Seurat::FindClusters function using the Louvain algorithm were used to cluster cells according to their batch corrected transcriptomes similarities. To visualize the cells similarities in a 2-dimension space, the Seurat::RunUMAP function using default parameter was used. Cell clusters were then annotated based on cell type specific gene expression markers.