Extended Data Fig. 1: Supplementary data for the isolation of target barcoded cells from a population using CloneSelect C → T.
From: A multi-kingdom genetic barcoding system for precise clone isolation
a, Schematic diagram for the barcode library construction. The ∆EGFP fragment (GTG mutated start codon) was amplified by PCR using pooled forward primers encoding the PAM followed by semi-random barcode sequences encoding the GTG mutated start codon and a common reverse primer. The PCR product was then enzymatically digested and ligated to the lentivirus plasmid backbone. b, The PCR product. c, Estimated complexities of the generated plasmid pools by colony forming units (n = 3). d, Library QC by single colony isolation followed by plasmid purification and restriction digestion using BsrGI, ClaI and PvuI. e, Sanger sequencing of the barcode region of the colony isolates. f and g, Barcode distribution in the lentiviral plasmid DNA pool and that in the cell population transduced using the same plasmid DNA pool. h, Barcode distributions of the EGFP-positive cell samples obtained by cell sorting after barcode-specific activation. The results of the 16 independent samples were combined after read count normalization applied to each sample. i, Frequency of the GTG → ATG mutation observed for each barcode after sorting the EGFP-positive cells. Each row represents the GTG → ATG mutation frequency profile in each target isolation assay.
