CRISPR sequencing libraries can be sequenced together or separately from Whole Transcriptome (WT) sequencing libraries. We recommend sequencing CRISPR libraries at 2,000-5,000 reads per cell, and WT libraries at 20,000 reads per cell as a starting point (see this article for more information on WT sequencing depth considerations). Therefore, the ratio between the libraries may need to be adjusted if they are sequenced together.
CRISPR and WT libraries require a 150 cycle kit minimum (which will get you up to 168 total cycles as Illumina includes extra reagents as a standard practice). For CRISPR libraries sequenced together with WT libraries, we recommend using a 5% PhiX spike in. If the CRISPR libraries are sequenced alone, we suggest using a minimum of 20% PhiX due to the limited complexity and this may need to be adjusted further based on the sequencing instrument. CRISPR libraries contain 5’ Nextera and 3’ Truseq adapters, and the UDI Plate - EC adds Nextera read 1 and Truseq read 2. WT libraries contain 5’ and 3’ TruSeq adapters, and the UDI Plate - WT adds TruSeq read 1 and Truseq read 2.
All Evercode sequencing libraries should be diluted and denatured according to the instruction for the relevant sequencing instrument. The run configuration requirements for WT and CRISPR Detect sequencing libraries is as follows and paired end sequencing should be used:
| Read | Cycles |
| Read 1 | 66 |
| i7 Index (Index 1) | 8 |
| i5 Index (Index 2) | 8 |
| Read 2 | 86 |
CRISPR sequencing amplicon
The guide sequence is depicted in gray (between Human U6 Primer 2 and BC1). A final PCR amplifies the CRISPR PCR product and appends the fourth DNA barcodes as UDIs in the UDI Plate - EC. This also adds P5 and P7 adaptors.
WT sequencing amplicon
The WT sequence is depicted in gray (between R1 and BC1). A final PCR amplifies the fragmented cDNA and appends the fourth DNA barcode, UDIs from the UDI Plate - WT, as well as the P5 and P7 adaptors.
Additional resources
For WT sequencing requirements see: What are the run configuration and sequencing requirements for WT libraries?
For UDI sequences see: Unique Dual Indices (UDI) Sequences