Chromium next gem single cell 3 v3

sgRNAs targeting the top hits from the CRISPRi screens and also candidate regulators selected based on literature were cloned into pMK1334. The concentration of each sgRNA plasmid was measured using the Qubit dsDNA HS Assay Kit on a Qubit 2.0 Fluorometer, and then the plasmids were pooled. iAstrocytes were transduced with lentivirus generated from the sgRNA pool at low multiplicity of infection so that <30% of cells were transduced, treated with vehicle control or IL-1α+TNF+C1q for 24 hours, and then sorted for sgRNA-transduced cells via FACS. Sorted iAstrocytes were then used as input for single-cell RNA-seq using Chromium Next GEM Single Cell 3’ v3.1 reagents (10X Genomics cat. no. PN-1000121, PN-1000127, and PN-1000213), loading ~45,000 cells per reaction into four reactions, with two reactions for vehicle control-treated iAstrocytes and two reactions for IL-1α+TNF+C1q-treated iAstrocytes. To facilitate association of single-cell transcriptomes with sgRNAs, sgRNA-containing transcripts were amplified as described in Tian et al.⁸⁵ . The sgRNA-enrichment libraries were separately indexed and sequenced as spike-ins alongside the whole-transcriptome single-cell RNA-seq libraries on a NovaSeq 6000, recovering on average ~29,000 transcriptome reads and ~5,000 sgRNA-containing transcript reads per cell.

For droplet-based snRNA-seq, libraries were prepared using the Chromium Next GEM Single Cell 3′ v.3.1 according to the manufacturer’s protocol (10x Genomics), targeting 10,000 nuclei per sample after counting with a TC20 Automated Cell Counter (Bio-Rad). Thirteen cycles were applied to brain parenchyma samples to generate cDNA, and 15 for choroid plexus samples. All samples underwent 15 or 16 cycles for final library generation. Generated snRNA-seq libraries were sequenced across two S4 lanes on a NovaSeq 6000 (150 cycles, Novogene).