title: GSM8491223: TF overexpression NGN2 iNeurons, batch 1, 409B2 genotype; Homo sapiens; RNA-Seq
library_construction_protocol: For ParseBio, to obtain single cell suspension, induced neurons cultured in 12-well plates were washed with PBS and incubated with 300 μL accutase at 37°C for 5 min. Dissociation reaction were stopped with 700 μL PBS containing 0.04% BSA, and cells were gently trituated into single cell suspension. Cells were washed twice with 500 μL PBS containing 0.04% BSA before nuclei isolation. To isolate the nuclei from single cell suspension, cells were trituated ten times in 100 μL lysis buffer (10 mM Tris-Cl pH7.4, 3 mM NaCl, 10 mM MgCl2, 1% BSA, 0.1% Tween-20, 1 mM DTT, 0.2U/μL RNase inhibitor (Takara), 0.1% NP-40, 0.01% Digitonin (Invitrogen)) and incubate 2 min on ice. The isolated nuclei underwent two subsequent washes in 500 μL wash buffer (10 mM Tris-Cl pH7.4, 3 mM NaCl, 10 mM MgCl2, 1% BSA, 0.1% Tween-20, 1 mM DTT, 0.2U/μL RNase inhibitor). Isolated nuclei were fixed and cryopreserved for snRNA-seq according to the instructions provided by the manufacturer (Parse Biosciences). For 10x Multiome, single-cell suspensions obtained from each NGN2-iN pre-patterning condition were divided into two distinct pools (Supplementary Table 1). Subsequently, each pool, along with the control NGN2-inducible iPSCs, underwent nuclei isolation individually following provider's instruction (10x Genomics user guide CG000365). For bulk RNA-seq, to extract RNA, cells were individually collected and lysed in Trizol (Invitrogen). Subsequently, RNA extraction was performed in the multi-well format using the MagMAX™-96 for microarrays total RNA isolation kit (Invitrogen). Quality assessment of the isolated RNAs from each condition was conducted using the RNA 6000 Pico Kit and the 2100 Bioanalyzer Systems (Agilent), while quantification was carried out using the Qubit system (Invitrogen). cDNA reverse transcription and amplification were carried out according to the smart-seq2 protocol (64). The preparation of the sequencing library was based on the Nextera XT DNA Library Prep protocol (Illumina), and subsequently sequenced using the Illumina NextSeq High Output paired-end 2x75 parameter (NGN2 pre-patterned progenitors) or NovaSeq SP paired-end 2x51 (reproducibility, stability and ASCL1-DLX2 pre-patterned progenitors). Sequencing reads from the fastq file were aligned against the hg38 reference genome (obtained from 10x Genomics) using the STAR RNA-seq aligner with default parameters. FACS sorted NGN2-iNs + TF-OE were pooled for GEM generation and cDNA preparation according to the 10x-Chromium Next GEM Single Cell 3' v3.1 (Dual Index) protocol. The resulting cDNA was split for separate transcriptome and feature barcode library preparation and pooled for sequencing on the Illumina NovaSeq 6000 platform using 28/10/10/90 as cycle parameters. FACS sorted TF-KO NGN2-iNs from the same morphogen condition (RA or BMP4) and their respective non-targeting control were pooled for scRNA-seq + CRISPR gRNA detection following the Chromium Next GEM Single Cell 5' v2 (Dual Index) kit protocol. The transcriptome libraries and gRNA libraries were pooled and sequenced on the NovaSeq 6000 platform using 26/10/10/90 as cycle parameters. For sample demultiplexing and generation of count matrices, cellranger -multi (10x Genomics) was utilized. To facilitate efficient gRNA detection, the pattern and sequence entries in the feature reference files were adjusted to 12bp. For gRNA label assignment, the 10x gRNA calling outputs (protospacer_calls_per_cell.csv) were further filtered to retain only cells with gRNAs targeting a single gene. Cells lacking gRNAs or those with gRNAs targeting multiple genes were excluded from further analysis. Cells with effective perturbations were identified using Mixscape (PMID: 33649593) as implemented in Seurat (PMID: 34062119). To visualize the enrichment or depletion of KO cells relative to controls, the densities of both control and KO cells on the UMAP embedding were estimated using Nebulosa (PMID: 33459785) with weight=1. KO cell densities were matched to the nearest control cells based on UMAP coordinates. The difference in density between the KO and corresponding control cells was then calculated and visualized on the UMAP embedding. Libraries were constructed according to the manufacter's instructions. For 10x Genomics user guide CG000338 was used.