Nucleospin gel and pcr clean up columns

Genomic DNA was isolated using the DNeasy Blood and Tissue Isolation Kit (QIAGEN). For melanocyte samples, the gDNA was cleaned of melanin using the OneStep^™ PCR Inhibitor Removal Kit (Zymo). To isolate the CRISPR guide cassette from the genome, PCR was done at one reaction for every 5×10⁵ cells using PrimeSTAR Max DNA Polymerase Premix (Takara). At this time, a unique 9-mer barcode was added to each guide cassette amplicon for removal of PCR duplicates (Table S7). Subsequent to 5 cycles of PCR, the reactions were pooled over NucleoSpin Gel and PCR Clean-Up Columns (Takara). A second PCR of 23-25 cycles was done to prepare the DNA for flow cell binding, including barcoding for multiplexing, and sufficient amplification. Standard Illumina i5/i7 barcodes were used. This round of PCR was followed by PCR cleanup over a single column and run on a 2.5% agarose gel to isolate the single product band for high-throughput sequencing. When necessitated by Bioanalyzer trace results, a secondary PAGE purification was carried out via standard protocol and purified over PCR cleanup columns. Samples were sequenced to a minimum average per unique guide coverage of 100× on the NextSeq Platform.