Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet: Technical performance and quality control of in situ sequencing by synthesis (SBS). Data are from a screen in A549 cells with a CROPseq-puro library of 5,738 sgRNAs43. (a) Example compensation matrix used for correcting spectral cross-talk between SBS imaging channels. (b) Spectral compensation of the two-channel combinations with the most cross-talk (T vs G and C vs A) at the first and last cycle of an SBS experiment. Mapped reads are those with barcode sequences exactly matching expected sequences from the designed sgRNA library. Dotted lines in the compensated plots demarcate the decision boundary for base calling. (c) Plotting read mapping rate and mapped reads per cell against increasing thresholds on the peak parameter demonstrate that most non-mapping reads are excluded by thresholding this value. (d) Longer read lengths provide increased confidence of mapped reads representing true sequencing spots from barcode mRNA. Plotting plate heatmaps of quality control metrics such as read mapping rate (e), total cells imaged (f), and fraction of cells with reads mapping to one expected barcode sequence (g) is useful for evaluating the quality of an experiment and identifying potential issues.

Article Snippet: NGS validation primers for CROPseq-puro vector (Integrated DNA technologies) NGS_CROPseq-puro_P5: ACACGACGCTCTTCCGATCTtcttgtggaaaggacgaaacNGS_CROPseq-puro_P7: CTGGAGTTCAGACGTGTGCTCTTCCGATCTaagcaccgactcggtgccac.

Techniques: In Situ, Sequencing, Imaging

Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet: Overview of optical pooled screening. (a) Experimental workflow. First, a pooled sgRNA library is designed, packaged into lentivirus, and delivered into Cas9-expressing cells. A live-cell or fixed-cell imaging assay is performed to generate an optical phenotypic profile of individual cells. The sgRNA spacer sequences in each cell are then amplified and read out by in situ sequencing by synthesis (SBS), consisting of cycles of dye incorporation, imaging, and cleavage. Finally, sgRNA-encoded perturbations are mapped to phenotypic scores at the single-cell level, with candidate genes identified through various statistical approaches. (b) Schematic of the in situ SBS process. The sgRNA is expressed as a polyadenylated mRNA transcript from an integrated copy of the CROPseq vector. After fixation and permeabilization, a locked nucleic acid (LNA)-modified primer is used to reverse transcribe a cDNA copy of the sgRNA sequence. After glutaraldehyde and formaldehyde post-fixation, the mRNA is digested and a padlock probe is hybridized to cDNA regions flanking the sgRNA sequence. The padlock probe is then extended and ligated to copy the sgRNA sequence into a single-stranded circularized DNA. This circularized DNA serves as a template for rolling circle amplification with Phi29 polymerase, the amplified product of which contains tandem repeats of the sgRNA spacer sequence. These sequences are read out by successive cycles of SBS.

Article Snippet: NGS validation primers for CROPseq-puro vector (Integrated DNA technologies) NGS_CROPseq-puro_P5: ACACGACGCTCTTCCGATCTtcttgtggaaaggacgaaacNGS_CROPseq-puro_P7: CTGGAGTTCAGACGTGTGCTCTTCCGATCTaagcaccgactcggtgccac.

Techniques: Expressing, Imaging, Amplification, In Situ, Sequencing, Plasmid Preparation, Modification

Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet: Troubleshooting table

Article Snippet: NGS validation primers for CROPseq-puro vector (Integrated DNA technologies) NGS_CROPseq-puro_P5: ACACGACGCTCTTCCGATCTtcttgtggaaaggacgaaacNGS_CROPseq-puro_P7: CTGGAGTTCAGACGTGTGCTCTTCCGATCTaagcaccgactcggtgccac.

Techniques: Transformation Assay, Electroporation, Plasmid Preparation, Amplification, Transfection, Transduction, Fluorescence, Imaging, Flow Cytometry, Expressing, Transferring, In Situ, Sequencing, Evaporation, Incubation, Buffer Exchange, Staining, Diffusion-based Assay, Selection, Infection, Concentration Assay, Microscopy, Software

Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet:

Article Snippet: NGS validation primers for CROPseq-puro vector (Integrated DNA technologies) NGS_CROPseq-puro_P5: ACACGACGCTCTTCCGATCTtcttgtggaaaggacgaaacNGS_CROPseq-puro_P7: CTGGAGTTCAGACGTGTGCTCTTCCGATCTaagcaccgactcggtgccac.

Techniques: Concentration Assay, Plasmid Preparation

Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet:

Article Snippet: NGS validation primers for CROPseq-puro vector (Integrated DNA technologies) NGS_CROPseq-puro_P5: ACACGACGCTCTTCCGATCTtcttgtggaaaggacgaaacNGS_CROPseq-puro_P7: CTGGAGTTCAGACGTGTGCTCTTCCGATCTaagcaccgactcggtgccac.

Techniques: Concentration Assay

Journal: Biology Methods & Protocols

Article Title: Fine-mapping within eQTL credible intervals by expression CROP-seq

doi: 10.1093/biomethods/bpaa008

Figure Lengend Snippet: Experimental design of expression CROP-seq screening of eSNPs. ( A ) SNPs were selected with various eQTL P -values from one or two credible intervals for each eGene. Additional SNPs in low LD with the credible interval were selected as control SNPs. Each SNP was targeted by a single gRNA with minimal predicted off-target effect. The horizontal black line represents a hypothetical locus with exons indicated by solid blocks. ( B ) The Cas9 editing site may be a few bases away from the targeted SNP and can introduce four possible genetic alterations: deletion of both the cutting site and target SNP; insertion; deletion of only the cutting site; and mutation of the target SNP. ( C ) Pooled CROP-seq lentiviral libraries with 67 gRNA were transduced into the HL60/S4 cell line. Most cells were transduced with a single gRNA. Red, green, yellow, and blue represent four different gRNAs. A few cells have zero (gray cell) or multiple gRNAs. After 10X single-cell RNA-seq identified the gRNA of each cell, differential expression of the linked transcript is evaluated between cells with the gRNA relative to cells with all other gRNAs.

Article Snippet: Lentivirus production from lentiviral vectors CROPseq-Guide-Puro and lentiCas9-Blast [ ] (Addgene, 52962) and was performed following Addgene’s standard lentivirus production protocol using the Lenti-X 293 T cell line (Takara, Kusatsu Japan, catalog number #632180).

Techniques: Expressing, Control, Introduce, Mutagenesis, Transduction, RNA Sequencing, Quantitative Proteomics