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.
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 sequencing libraries depend on if the libraries were prepared using CRISPR Detect for Evercode WT v2 or CRISPR Detect for WT v3. Please see the user guide used to prepare the libraries (linked below) for the run configuration requirements.