Concentrate kit

Plasmids were isolated from bacterial pellets by miniprep (Qiagen). Illumina sequencing regions were added to either side of the eCPX-peptide gene by PCR amplification using Q5 DNA polymerase (New England Biolabs) following the manufacturer's protocol and primers 1-F and 1-R (see Figure S2) PCR products were cleaned by gel extraction and re-concentrated using a ZymoClean Clean & Concentrate kit. Another PCR amplification was performed also using Q5 to add the P5 and P7 Illumina sequences for flow cell annealing as well as a unique 8 letter barcode on each end of the amplicon for demultiplexing using primers 5-(0-7) and 7-(0-7). 38 The second round of PCR was cleaned and re-concentrated identically. DNA concentrations were quantified using a QuBit fluorimeter, pooled, and submitted to the University of Michigan DNA Advanced Genomics core for analysis. Samples were demultiplexed by sorting samples with perfectly matched barcodes and ones that differed by up to one base pair. Samples were then analyzed with FastQC and samples with a PHRED score of less than 36 were discarded. Fastq files were then analyzed using Python scripts with Biopython and SeqIO packages. Forward and reverse reads were pairwise analyzed, discarding any sequences with differences in base pairs. The portion of the read that corresponds to the p53-based peptide was translated.