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Get an NGS Analysis kit that has all the primers and reagents to prepare sgRNA, shRNA, or barcodes from pooled lentiviral libraries for sequencing, or send Cellecta your samples and we will prep and sequence them for you.
Pooled lentiviral CRISPR, shRNA, and barcode libraries require targeted PCR amplification of the effectors from genomic DNA after screens. Cellecta offers kits with complete reagents and primers to generate sequencing-ready samples from genomic DNA isolated from pooled cells. Software included with the kit enables rapid and convenient analysis of the sequencing.The program demultiplexes indexed samples and aligns sgRNA reads from the Illumina NGS FASTQ files. The output is a spreadsheet with the counts of each sgRNA in each sample.
Alternatively, we provide complete Next-Gen Sequencing (NGS) and analysis services for researchers running their own screens or cell tracking experiments with pooled libraries.
Use of a pooled lentiviral CRISPR, shRNA, or barcode library in an experiment requires amplification and next-generation sequencing (NGS) analysis of the barcodes or guides from genomic DNA isolated from cells transduced with the library. Our service includes the DNA preparation, sequencing run, and demultiplexing/alignment, so you receive a spreadsheet with reads/guide/sample for each element in the library, with optional DNA isolation from cells or tissue samples.
Cellecta NGS Analysis Kits provide the enzymes, reagents, and 15 indexed amplification primers used to prepare sequencing libraries for multiplex NGS analysis. Primers are specific to each library/vector combination, so you simply need to choose a kit appropriate for the library you are using. Each kit contains sufficient primers and reagents for 6-48 samples (48 preps of 50 ug each, 12 multiplex). Supplementary indexing primers are also available, which increase multiplexing capacity to 24 samples.
Alternatively, you can send Cellecta cells, tissues, or genomic DNA and have us prepare, sequence and analyze the samples for you.
After running screens with pooled lentiviral CRISPR, shRNA, or tracking cells using barcode libraries, genomic DNA from the whole transduced population of cells is isolated, and the frequency of each integrated shRNA, sgRNA, or barcode lentiviral construct in the population is assessed by next-generation sequencing (NGS).
For CRISPR and shRNA screens, the frequency of each guide or hairpin is assessed to determined which ones are enriched or depleted relative to the original libraries or control samples. For example, “dropout viability” screens may be run to identify sgRNA or shRNA that are toxic to cells because they target essential genes. This sort of screen is often used to look for genetic susceptibilities in cancer cells.
With barcoding studies, cells labeled by transduction of complex pooled libraries of unique DNA sequences are similarly sequenced to assess the diversity and quantities of each of the barcodes present in the cell population.
