Single-nucleus RNA sequencing of hiPSC-derived neurons generated by different methods

Single-nucleus RNA sequencing of hiPSC-derived neurons (hiNs) generated by three different methods:
1) Direct hiPSC-to-neuron lineage reprogramming using Neurog2 (4 weeks)
2) Neural induction by dual SMAD-inhibition followed by spontaneous neuronal differentiation (4 and 6 weeks)
3) Ascl1 expression in neural progenitor cells (4 weeks)

HiNs cultures at 4 or 6 weeks were washed in the imaging plate wells with 500 µL of Deionized Phosphate Buffer Saline 0.1M (DPBS, GIBCO™, Fisher Scientific 11590476). Cells were resuspended in 1 ml of 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 10 mins. at 500 g and 4°C (used for all following centrifugation steps). Nuclei pellets were washed three times with one filtration step by MACS pre-separation filter 30μm (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. Sample cell-hashing was performed using 0.5 μL of Total-Seq™antibodies (Biolegend) and incubation for additional 15 min at 4°C. Nuclei pellets were washed three times in staining buffer with one filtration step by MACS pre-separation filter 30 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, Fisher Scientific). 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 6000system following the manufacturer recommendations (Illumina).