Journal: bioRxiv

Article Title: High complexity cellular barcoding and clonal tracing reveals stochastic and deterministic parameters of radiation resistance

doi: 10.1101/2020.10.13.337519

Figure Lengend Snippet: ( a ) Bar plots showing the number of detected barcodes in the initial starting population of FaDu, HNO407 and HNO206 after transduction with the ClonTracer lentiviral barcode library, antibiotic selection, expansion and bioinformatic processing. One technical replicate of initial starting population was processed for the FaDu line and two technical replicates for the HNO407 and HNO206 line. ( b ) Distribution of the barcode sequencing reads (log 2 ) of the initial starting population in FaDu and of two technical replicates of the initial starting populations of HNO407 and HNO206. ( c ) Venn diagram displaying the number of overlapping barcodes found in each initial starting population of HNO407 and HNO206. All detected barcodes after bioinformatic processing were included. Tables show percentages of shared and non-shared barcodes among the two processed initial starting populations.

Article Snippet: For each 10cm dish, 2.4μg ClonTracer plasmid DNA, 2.4μg psPAX2 (encoding HIV-1 Gag, Pol, Tat and Rev proteins) and 0.6μg pMD2.G (encoding VSV G envelope protein) was diluted in 2000μl Opti-MEM ® Reduced Serum medium (Gibco™/ Life Technologies).

Techniques: Transduction, Selection, Sequencing

Journal: bioRxiv

Article Title: High complexity cellular barcoding and clonal tracing reveals stochastic and deterministic parameters of radiation resistance

doi: 10.1101/2020.10.13.337519

Figure Lengend Snippet: A Experimental overview. Upon transduction of the HNSCC cell lines with the lentiviral ClonTracer barcode library, cells were expanded and fractionated irradiated with 5 fractions of 4Gy or maintained under normal non-irradiated conditions. 48 h after last irradiation cells were seeded in multiple 150 mm2 dishes in low density for colony formation. Colonies were harvested as a clonal pool and barcode sequences were PCR-amplified from genomic DNA (gDNA) and subjected to NGS. NGS data was processed by in-house software for subsequent analysis of clonal dynamics. B Bar plots showing the average proportion of detected barcodes after colony formation in nonirradiated (Ctr, n=3) and irradiated (IR, n=4) samples in FaDu (gray), HNO407 (red) and HNO206 (green). Percentages are calculated relative to the number of barcodes detected in the initial starting population of the respective cell line (***, p<0.0001, error bars = SD). C Cumulative sum of barcode sequencing reads of non-irradiated (blue) and irradiated (red) samples in FaDu, HNO407 and HNO206. The average number of barcodes that constitute 95% of total barcode sequencing reads under non-irradiated and irradiated conditions is highlighted. D Bars demonstrate the average proportion of detected barcodes in control (Ctr, blue) and irradiated replicates (IR, red) that contribute to 95% of the total barcode sequencing reads. E Numerical modeling of the number of detected barcodes after each fraction of radiation in FaDu, HNO407 and HNO206 assuming that all cell clones have the same radiation survival probability and the same capability to repopulate after each treatment cycle. F Bar plots showing the average model-predicted proportion of detected barcodes after 5 fractions of photon radiation (4Gy) and experimentally detected barcodes in FaDu (gray), HNO407 (red) and HNO206 (green), (n =4; ***, p<0.0001, error bars = SD).

Article Snippet: For each 10cm dish, 2.4μg ClonTracer plasmid DNA, 2.4μg psPAX2 (encoding HIV-1 Gag, Pol, Tat and Rev proteins) and 0.6μg pMD2.G (encoding VSV G envelope protein) was diluted in 2000μl Opti-MEM ® Reduced Serum medium (Gibco™/ Life Technologies).

Techniques: Transduction, Irradiation, Amplification, Software, Sequencing, Clone Assay