Step 1: Design your CRISPR library
Pick the target genes whose function you'd like to further investigate—from a deep dive into a focused set of tens of genes to a broad screen of hundreds of genes that comprise many pathways. Then, design your sgRNAs to order, with one of many freely available CRISPR guide RNA design tools, to clone into a 10x Genomics capture sequence enabled CRISPR vector.
Step 2: Prepare your Sample
Transduce and stimulate your cells—infect your cells, select the cells expressing your CRISPR guide, and apply your stimulus. Harvest your cells, and start exploring across tens of thousands of cells. Follow best practices for washing, counting, and concentrating cells to minimise the presence of cellular aggregates, and retrieve high quality single cell suspensions.
Step 3: Construct your 10x Library
Construct a 10x barcoded library using their reagent kits and Chromium Controller. The Chromium Controller encapsulates each cell with a 10x barcoded Gel Bead in a single partition. Within each nanoliter-scale partition, cells undergo reverse transcription to generate cDNA for both mRNA and CRISPR guides, each of which shares a 10x Barcode with all cDNA from its individual cell of origin.
Step 4: Sequence
Sequence the resulting 10x barcoded library on compatible standard NGS short-read sequencers for massive transcriptional profiling of thousands of individual cells.
Step 5: Analyse Your Data
Use the 10x Cell Ranger analysis software to automatically assign guides on a per cell basis and directly assess perturbation effects of a guide on the target gene as well as the entire transcriptome.