Supplementary Figure 1: Optimization of modified of guide constant regions to enable 3’ direct capture Perturb-seq.

a) Schematic of 3’ single-cell RNA-sequencing (3’ scRNA-seq). Polyadenylated mRNAs from individual cells (top, light blue) anneal to barcoded oligo-dT primers in emulsion droplets (delivered to droplets on gel beads) and are reverse transcribed into indexed cDNA (bottom). TSO, template switch oligo. UMI, unique molecular identifier. CBC, cell barcode. b) Schematic of 5’ single-cell RNA-sequencing (5’ scRNA-seq). Polyadenylated mRNAs from individual cells (top, light blue) anneal to unbarcoded oligo-dT primers in emulsion droplets (delivered to droplets as free oligos) and are reverse transcribed. Indexing of cDNA (bottom) occurs when template switching allows for extension of barcoded TSOs (delivered to droplets on gel beads). c) Schematic of constant region 1 (CR1) guide RNAs. Arrows indicate the positions of capture sequence insertions. d) CRISPRi activity of guides carrying the indicated capture sequences (all programmed with an identical GFP targeting sequence) in GFP+ K562 dCas9-KRAB cells 10 days post-transduction. Data from guides selected for direct capture experiments (sgRNA-CR1^cs1 and sgRNA-CR1^cs2) are indicated. For comparison, data from standard guides targeting GFP (programmed with the same targeting region but without capture sequences and expressed from 3 other vectors) were also included. One of these, indicated as “CROP-seq”, has a different, previously published (Datlinger et al., Nature Methods, 14-3, 2017)⁸ constant region and is expressed from a different promoter. Data represents the average of independently infected triplicates normalized to controls ± standard deviation. The data was collected in two separate batches (independently controlled). Representative flow cytometry gating for one sample is also shown. e) Gaussian kernel density estimates of normalized flow-cytometry measurements representing GFP expression demonstrate CRISPRi activity of the indicated sgRNAs (programmed with an identical GFP targeting sequence). Data was collected in three independent biological replicates and a representative replicate is shown. AU, arbitrary units. f) Gaussian kernel density estimates of normalized flow-cytometry measurements representing GFP expression demonstrate CRISPRi activity of the indicated sgRNAs (programmed with an identical GFP targeting region). Data was collected in three independent biological replicates and a representative replicate is shown. AU, arbitrary units. g) Schematic of experimental workflow for direct capture Perturb-seq (3’ or 5’) based on protocols from 10x Genomics. Red indicates generation of sequencing libraries. Box details construction of index sequencing library for GBC Perturb-seq, which is based on a previously published protocol (Adamson et al., Cell, 167-7, 2016)⁴.