Question: What sequencing length should I use for Read 2 of my 5’ CRISPR library?
Answer: For in-house sequencing of Single Cell 5’ CRISPR libraries, we pool 5’ CRISPR libraries with 5’ Gene Expression libraries and sequence Read 2 to a length of 90 bp. This read length is suitable for the sgRNA backbones we tested in-house. However, if a different sgRNA backbone is used that is longer than the backbone we tested in-house, it is possible that a longer Read 2 sequencing length may be required.
When sequencing the 5’ CRISPR library, the Read 2 sequence will be the reverse complement sequence, starting at the region complementary to the CRISPR RT primer (“Feature Primer” in the diagram below), and reading toward the protospacer (“Feature Barcode” in the diagram below). Refer to the library structure in the appropriate 5’ User Guide for further details.
To ensure that the full protospacer is sequenced, Read 2 needs to be long enough to read through the Feature Primer, sgRNA backbone and protospacer. When calculating a minimum Read 2 length, we recommend calculating the length from the Feature Primer to the Feature Barcode (protospacer).
Below is an example of an sgRNA we tested in-house. The protospacer is shown in purple. The Feature Primer is shown in blue. The length from the protospacer to Feature Primer is 83 nucleotides. Thus, for this sgRNA backbone, a Read 2 length of 83 bp or longer would be sufficient to read the full protospacer sequence.
- NNNNNNNNNNNNNNNNNNNNGTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT
If you are using an sgRNA backbone for which the distance from the protospacer to Feature Primer is more than 90 nucleotides, a Read 2 length greater than 90 bp would be needed to sequence the full protospacer sequence.
Product: Single Cell Immune Profiling