Protocols #

Cloning and Plasmid Preparation #

If you want to work with a pre-built GuEST-List library (such as the genome-wide CRISPRi library from our preprint)…

• You can order the plasmid from Addgene
• You can follow our protocol for amplifying the library from a small aliquot of plasmid (PDF Download)

If you want to build your own VIPerturb-seq compatible guide library…

• You can adapt our protocol for cloning into our custom CROPseq vector to whichever version of the vector is best-suited for your system (.gbk sequence Download) (PDF Download)
• You can find a list of recommended barcodes to use on our Sequences Page

We also recommend following our protocols for generating bulk sequencing libraries from GuEST-List plasmid to verify library diversity after sequencing. You can find the protocol for that here and some code to analyze the resulting data here.

Running VIPerturb-seq #

Here we provide two sample workflows for conducting single-cell screens and preparing the resulting libraries with 10x Genomics.

• VIPerturb-seq Workflow for Phenotypic Enrichment on Intracellular Protein with Flex v1 (PDF)
• Multimodal VIPerturb-seq Workflow for capturing whole transcriptome, GuEST-List guide barcodes, surface and/or intracellular protein, and hashing identity from Flex v1 workflows (PDF) (original FlexPlex citation)