title: GSM6598989: LUMC04i10 and WA01 D110; Homo sapiens; RNA-Seq
library_construction_protocol: Aggregate dissociation to single cells for scRNAseq    Vibratomed sections or randomly selected aggregates were pooled at specified time points. Three to four number of aggregates were placed in warm TrypLE (12605036, Gibco) and incubated in a 37°C incubator under 5% CO2 on an orbital shaker set to 65 rpm. The mixture was triturated with a pipette using wide bore p1000 tips every 10 minutes. Dissociation to single cells was complete after 60 minutes and confirmed by bright-field visualization using a Nikon TS2R. TrypLE activity was stopped by adding cold 3% bovine serum albumin (BSA; A7030, Sigma‐Aldrich) in PBS. To remove residual cell aggregates and debris, the suspension was filtered with a 40 μm Flowmi cell strainer (H13680-0040, Bel-Art). After washing three times with 3% BSA in PBS, cells were resuspended in cold 3% BSA in PBS and filtered again with a 40 μm Flowmi cell strainer. Cell viability was determined by using Trypan Blue (15250061, Gibco) and an automated cell counter (Countess II, Thermo Fisher). Cell suspensions were diluted with cold 3% BSA in PBS to reach a final concentration of 1,000 cells/μL.     Aggregate dissociation to single nuclei for snRNAseq  2-4 randomly selected aggregates were processed at specified time points. Aggregates were pooled, washed once with ice-cold PBS, and resuspended in lysis buffer that contained 10 mM Tris-HCl (T2194, Millipore-Sigma), 10 mM NaCl (59222C, Millipore-Sigma), 3 mM MgCl2 (M1028, Millipore-Sigma), 0.1% Nonidet P40 Substitute (74385, Millipore-Sigma) in DEPC-treated water (750024, Invitrogen). The suspension was transferred to a Dounce tissue grinder (885300-0002, Kimble) and ground every 5 min. After 20 min, dissociation to single nuclei was confirmed by bright-field visualization (EVOS M5000, Thermo Scientific). After trituration using p1000 and p200 tips, the suspension was filtered through a 70 μm MACS SmartStrainer (130-098-462, Miltenyi Biotec), centrifuged at 500xg for 5 min at 4°C and resuspended in 1% BSA in PBS (AM2616, Invitrogen) containing 200 U/μL RNase inhibitor (3335402001, Millipore-Sigma). After filtration using a 40 μm Flowmi cell strainer and centrifugation at 500g for 5 minutes at 4°C, the nuclei suspension was resuspended to a final concentration of 1,000 cells/μL.  For one experiment of D110 aggregates generated using the LUMC04i10 hiPSC line, we performed nuclear hashtagging of two separate aggregates using single nuclei hashing antibodies (MAb414, Merck). To this end, the nuclei suspension was blocked using Fc Blocking reagent (422302, Biolegend) for 5 min on ice followed by incubation with the hashing antibody (1 µg per 100 µl) for 10 min on ice. Subsequently, nuclei were washed three times using 1% BSA in PBS containing 200 U/μL RNase inhibitor, pooled together 1:1, and resuspended to a final concentration of 1,000 cells/μL. Single cell 3' RNA-seq experiments were conducted using the Chromium single cell system (10x Genomics, Inc) and the NexSeq500 or NovaSeq 6000 sequencer (Illumina, Inc). Approximately 17,000 cells or nuclei (targeting for 10,000 cells) per sample were added to a single cell master mix, following the Chromium Single Cell 3' Reagent Kits v2 and v3.1 User Guide (10x Genomics, Inc). Along with the single-cell gel beads and partitioning oil in separate wells of a Single Cell A Chip or Chromium Next GEM Chip G Single Cell (10x Genomics), the single cell reaction mixture was loaded to the Chromium Controller for GEM generation and barcoding, followed by cDNA synthesis and library preparation. At each step, the quality of cDNA and library was examined by Bioanalyzer. The resulting library was sequenced in a custom program for 26b plus 98b paired-end sequencing on an Illumina NextSeq500 or 28b plus 91b paired-end on Illumina NovaSeq 6000 to a read depth of >30,000 reads per cell. FASTQ files were generated using Cell Ranger mkfastq (10x Genomics).