Question: What workflow modifications are required when performing CRISPR Screening with Flex Gene Expression assays?
Answer: Workflow modifications to enable CRISPR Screening experiments are outlined in Technical Note CG000814 - CRISPR Screening with GEM-X Flex Gene Expression.
The guidance in Technical Note CG000814 is designed for the GEM-X Flex Gene Expression assay.
If performing CRISPR Screening experiments with Next GEM Flex Gene Expression assays, the guidance in CG000814 can be followed, with the following modifications to the “Protocol Modifications” section of CG000814:
- Step 1. Probe Hybridization
- The volumes listed in CG000814 should be modified, given that Next GEM Flex assays use a total volume of 100 μl for probe hybridization as compared to 50 μl for GEM-X Flex assays.
- For Next GEM assays, add 5 μl (instead of 2.5 μl) of the LHS & RHS CRISPR probes working stock to the Modified Probe Hybridization Mix.
- Follow the recommended volumes of Hyb Buffer B, Enhancer and Human/Mouse WTA probes in the relevant Next GEM Flex User Guide.
- Set the thermal cycler reaction volume to 100 μl.
- The volumes listed in CG000814 should be modified, given that Next GEM Flex assays use a total volume of 100 μl for probe hybridization as compared to 50 μl for GEM-X Flex assays.
- Step 4. Pre-Amplification PCR
- For Next GEM assays, use Amp Mix (PN-2000103) instead of Amp Mix C (PN-2001311).
- Primer choice for Next GEM assays:
- For Singleplexed (CG000691) and Multiplexed (CG000527) workflows, use either:
- Pre-Amp Primers B (P/N 2000529) supplemented with a user-supplied TruSeq R2 primer, or
- Feature Pre-Amp Primers (P/N 2000515).
- For Singleplex (CG000674) and Multiplex Feature Barcode technology for Protein using Barcode Oligo Capture (CG000673) workflows, use Pre-Amp Primers C (P/N 2000953) supplemented with an additional TruSeq Read 2 primer.
- For Singleplexed (CG000691) and Multiplexed (CG000527) workflows, use either:
For the Singleplexed with Feature Barcode technology for Protein (CG000477) workflow, use Feature Pre-Amp Primers (P/N 2000515).
While no impact on assay performance is anticipated, the use of custom probes in these assays is not officially supported by 10x Genomics. The additional guidance and resources that have been provided in this article are intended to help enable these experiments and improve customer success.
Products: Flex Gene Expression