Hifi 2x master mix

CRISPR/Cas9 mediated FORCP knockout LS180 cells were generated by Synthego Corporation (Redwood City, CA, USA). To generate KO cells, Ribonucleoproteins containing the Cas9 protein and synthetic chemically modified sgRNA were electroporated into LS180 cells using Synthego’s optimized protocol. Editing efficiency was assessed 48 hr post-electroporation by extracting genomic DNA from a pool of transfected cells followed by PCR amplification and Sanger sequencing. The resulting chromatograms were processed using Synthego Inference of CRISPR edits software (ice.synthego.com). The pool of cells was then seeded in 96-well plates at one cell per well followed by clonal selection and RT-qPCR to determine the effect on FORCP mRNA levels.
For indel analysis using Illumina sequencing, 20 ng of gDNA was used as template to amplify around the sgRNA target site using the primers listed. Briefly, two rounds of PCR were performed using the Kapa HiFi 2X mastermix in order to generate amplicons that can be sequenced using the Illumina MiSeq 2 × 150 format. Paired-end reads were generated, merged using FLASH (Magoč and Salzberg, 2011 (link)), filtered for quality, and subsequently mapped to the reference amplicon sequence using bwa mem. Sorted and indexed BAM files, generated by samtools, were then visualized using the Integrative Genomics Viewer (IGV).

All samples were amplified and prepared for sequencing using a one‐step PCR protocol as previously described³. More specifically, 1 μL of primers (5 uM for Final Reverse/Forward, 0.5 uM for Base Forward) were added to 5 μL of Kapa HiFi 2X master mix, and 4 μL template DNA/water. When the PCR reaction did not produce clear bands, the primer concentrations, DNA template input, PCR temperature, and number of cycles were optimized for individual samples.