Amplicon seq of BFP in BFP HEK293 cells transfected with NNG-Slug-nCas9-PolI5M and a gRNA targeting a nick 14 bp downstream of C 199 with respect to the sense strand of BFP cDNA: replicate 1
The BFP locus was amplified from 500 ng of gDNA for 20 cycles of PCR using oligonucleotides GCTCTTCCGATCTNNNNNACCCTGAAGTTCATCTGCACCA and GCTCTTCCGATCTNNNNNTTGAAGAAGATGGTGCGCTCCT. The PCR product was purified using PCR Reaction Cleanup beads from the UC Berkeley Sequencing Facility located in 310 Barker Hall, Berkeley, CA 94720. The beads were warmed to room temperature and mixed thoroughly in 9:5 volumetric ratio with the PCR product and incubated at room temperature for 5 minutes. Beads were separated from the solution by placing them on magnetic racks and washed with 70% ethanol twice. Ethanol was removed and beads were allowed to dry at room temperature for 30 minutes. The beads were removed from the magnetic rack and thoroughly resuspended in water to elute PCR 1 product. The concentration of PCR 1 product was quantified with a Qubit fluorometer using the high-sensitivity DNA kit. 10 ng of PCR 1 product was added to 10 additional cycles of PCR, in which Illumina adapter sequences and unique dual index pairs were attached. The molar concentration of each library prep was quantified by qPCR prior to pooling at equimolar amounts. The library was sequenced using an Illumina MiSeq V2 150 paired-end kit on an Illumina MiSeq sequencer. Samples were demultiplexed using bcl2fastq (v2.20).