D1000 screentape assay for tapestation systems

DNA was extracted from liver sections using Macherey-Nagel NucleoSpin Tissue Extraction Kit (Macherey-Nagel, REF 740952.250) with a final elution in 100 μl nuclease-free H2O. A 647 bp genomic region of the targeted locus was enriched by PCR before sequencing. Qubit quantitation (Invitrogen Qubit dsDNA HS and BR Assays) and TapeStation assay (Agilent Technology D1000 ScreenTape Assay for TapeStation Systems) were employed to quantify and determine the purity of the amplicon. Sequencing libraries were constructed following the NEBNext Ultra II (NEB) library preparation protocol. Sequencing was performed in a MiniSeq (Illumina) instrument with 2x150 paired-end reads at the Cima Genomics Platform (Pamplona, Spain).
The following pipeline was followed for the bioinformatic analysis of indel frequencies: Adapters and low-quality sequences were filtered with Trimmomatic [44 (link)]. Pair-end reads were merged with FLASH [45 (link)] and mapping to the mouse genome was performed with minimap2 (Li, 2018) with A5 -B4 -O25 -E1 options. Fragments mapping to a window of +/-100 bp around the cutting site were selected with samtools [46 (link)] and editing events were quantified with CrispRVariants R package [47 (link),48 (link)]. BioProject: PRJNA1090862. Link to the NGS data: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1090862.

DSP collection sample plates were dried, resuspended in nuclease-free water, and amplified using PCR according to the manufacturer’s protocol.
Following PCR amplification, the indexed libraries were pooled, harvested and washed using AMPure XP beads (Beckman Coulter) prior to elution. Sequencing library size was assessed with a High Sensitivity D1000 Screen Tape assay for TapeStation systems (Agilent Technologies) and the expected library size of ∼150 bp was observed. Purified libraries were sequenced by Illumina NovaSeq SP (2x 50bp).

Total genomic DNA was extracted from frozen liver sections using a NucleoSpin Tissue Extraction Kit (Macherey-Nagel, REF 740952.250) with a final elution in 100 µl nuclease-free H₂O and Qubit quantitation (Invitrogen™ Qubit™ dsDNA HS and BR Assays, Ref. #Q33230, Ref. #Q33261, respectively). On-target indel analysis was performed by deep sequencing and nested PCR was executed for NGS library preparation.
A first PCR round was performed by mixing 250 ng of template, Kapa HiFi HotStart ReadyMix PCR Kit (Roche, #KK2601), and primers (Hao1-int1 Fw and Hao1-int2 Rv; Appendix Table S1) that amplified 646 bp of the mouse Hao1 gene (containing the targeted region in the middle of the amplicon). The PCR product was purified using the Agencourt AMPure XP system (Beckman Coulter, #A63881) and eluted in 10 µl nuclease-free H₂O. Qubit quantitation (Invitrogen™ Qubit™ dsDNA HS and BR Assays) and TapeStation assay (Agilent Technology D1000 ScreenTape Assay for TapeStation Systems) were employed to quantify and determine the purity of the first amplicon before proceeding with the second PCR step.
Then, 40 ng of the PCR product was mixed with primers carrying partial universal adaptors for Illumina amplicons (Hao1-ex2 Fw and Hao1-ex2 Rv; Appendix Table S1). 450 bp amplicons were purified as described above and sent to GENEWIZ (GENEWIZ Germany GmbH) for Amplicon‐EZ analysis.