Amplicon seq of MAP2K1 in A375 cells transfected with EvolvR and on target gRNAs: forward reads replicate 2
A section of the MAP2K1 exon 6 locus was amplified from 360 ng of gDNA for 20 cycles of PCR using forward primer GCTCTTCCGATCTNNNNNCCCTCCTTTTCTATTTTCTCTTCCCTGCAG and reverse primer GCTCTTCCGATCTNNNNNCCGACATGTAGGACCTTGTGCCC. 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 applied to a second PCR, in which Illumina adapter sequences and unique dual index pairs were attached. The samples were delivered to the Innovative Genomic Institute Sequencing Core, where the samples the molar concentration of each library prep was quantified by qPCR prior to equimolar pooling. 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).