cell culture tf 1 cells  (ATCC)

Journal: Cell

Article Title: Lineage Tracing In Humans Enabled By Mitochondrial Mutations And Single Cell Genomics

doi: 10.1016/j.cell.2019.01.022

Figure Lengend Snippet: (A) Dynamics of mtDNA heteroplasmy in single cells. Each cell has multiple mitochondria, which in turn contain many copies of mtDNA that may acquire somatic mutations over time. (B) Proof-of-principle design. Each TF1 cell clone and sub-clone is assayed with ATAC-seq. (C) Supervised (true) experimental TF1 lineage tree. Colors indicate each primary clone from initial split. (D) Allelic heteroplasmy of four selected variants reveals stable propagation and clone-specificity. Color bar: allelic heteroplasmy (%). (E) Unsupervised hierarchical clustering of TF1 clones. Color: primary clones as in (C). (F) Between-clone and within-clone accuracy of identifying the most-recent common ancestor (MRCA) per trio of clones based on mtDNA mutational profile. (G) Schematic of mitochondrial relatedness matrix Kmito where each pair of clones is scored based on mitochondrial genotype similarity. (H) Random effects model for variance decomposition of epigenomic peaks. (I) Two examples of peaks inherited in clonal lineages. Peaks represent the sum of open chromatin for the clones with the most samples.

Article Snippet: EXPERIMENTAL MODEL AND SUBJECT DETAILS TF1 Cell Culture TF1 cells (ATCC) were maintained in Roswell Park Memorial Institute Medium (RPMI) 1640, 10% fetal bovine serum (FBS), 2mM L-Glutamine and 2ng/ml recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (Peprotech) and incubated at 37°C and 5% CO2.

Techniques: Clone Assay

Journal: Cell

Article Title: Lineage Tracing In Humans Enabled By Mitochondrial Mutations And Single Cell Genomics

doi: 10.1016/j.cell.2019.01.022

Figure Lengend Snippet: (A) Coverage of mouse mitochondrial genome by six scRNA-seq methods. Shown is the fraction (%) of the mitochondrial genome (y axis) covered by reads from each of six methods (color code), at different levels of coverage (x axis). (B) Agreement in allelic heteroplasmy estimates from single cell whole genome sequencing (WGS) and scRNA-seq from the same single cells. Shown is the allele frequency for scRNA- (y axis) and scWGS-seq (x axis) based estimates for two cell lines (HCC827: orange; SKBR3: purple). Two examples of RNA-specific changes are highlighted. (C-F) Identification of mitochondrial mutations by scRNA-, scATAC- and scMito-seq in three TF1 clones. (C) Bulk and single cell data collected for three TF1 clones (boxed). Each clone (n = 3) was processed with variable numbers of single-cell libraries (k). (D) Agreement in allelic heteroplasmy estimates from bulk ATAC- (x axis) and bulk RNA-seq (y axis) from three indicated TF1 clones (as in (C)). Two examples of RNA-specific changes are highlighted. (E) Coverage of the mitochondrial genome of the TF clone G10 by each indicated assay. Inner circle: mitochondrial genome; middle blue outline: coverage; outer grey circle: genome coordinates. For single cell assays, coverage is the sum of single cells. (F) Four clone-specific mutations that are reliably detected by various single-cell assays with heteroplasmies as low as 3.8%. Each boxplot shows the % heteroplasmy (y axis) of one mutation across scATAC-, scMito- and scRNA-seq in the three TF1 clones (color code as in (C)). Dots: individual cells.

Article Snippet: EXPERIMENTAL MODEL AND SUBJECT DETAILS TF1 Cell Culture TF1 cells (ATCC) were maintained in Roswell Park Memorial Institute Medium (RPMI) 1640, 10% fetal bovine serum (FBS), 2mM L-Glutamine and 2ng/ml recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (Peprotech) and incubated at 37°C and 5% CO2.

Techniques: Sequencing, Clone Assay, RNA Sequencing, Mutagenesis

Journal: Cell

Article Title: Lineage Tracing In Humans Enabled By Mitochondrial Mutations And Single Cell Genomics

doi: 10.1016/j.cell.2019.01.022

Figure Lengend Snippet: (A) Experimental overview. TF1 cells were infected with a lentiviral vector expressing the mNeonGreen gene and a 30bp random barcode in the untranslated region (Figure S3A). 25 cells were sorted and expanded, followed by bulk ATAC-seq and scRNA-seq. (B) Filtering of high confidence mutations. Base quality (BQ) scores from scRNA- (y axis) and from bulk ATAC-seq (x axis). White box: high-confidence variants detected by both technologies (BQ >20) (STAR Methods). (C) Allele frequencies determined by the sum of single cells from scRNA-seq (y axis) and bulk ATAC-seq (x axis). Black – filtered; red – retained. (D-F) mtDNA inferred clones agree with barcode-based clones. (D) Hierarchical clustering of TF1 mitochondrial genotyping profiles (rows) for cells assigned to annotated barcode groups (columns) (from Figure S3A). Color bar: Heteroplasmy (% allele frequency). (E) Cell-cell similarity from mitochondrial mutations called in (C). Column and rows are annotated by barcode group. (F) Between-group accuracy of identifying the most-similar pair per trio of clones based on mtDNA mutational profile using detected barcodes as a true positive.

Article Snippet: EXPERIMENTAL MODEL AND SUBJECT DETAILS TF1 Cell Culture TF1 cells (ATCC) were maintained in Roswell Park Memorial Institute Medium (RPMI) 1640, 10% fetal bovine serum (FBS), 2mM L-Glutamine and 2ng/ml recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (Peprotech) and incubated at 37°C and 5% CO2.

Techniques: Infection, Plasmid Preparation, Expressing, Clone Assay

Journal: Cell

Article Title: Lineage Tracing In Humans Enabled By Mitochondrial Mutations And Single Cell Genomics

doi: 10.1016/j.cell.2019.01.022

Figure Lengend Snippet: (A) Dynamics of mtDNA heteroplasmy in single cells. Each cell has multiple mitochondria, which in turn contain many copies of mtDNA that may acquire somatic mutations over time. (B) Proof-of-principle design. Each TF1 cell clone and sub-clone is assayed with ATAC-seq. (C) Supervised (true) experimental TF1 lineage tree. Colors indicate each primary clone from initial split. (D) Allelic heteroplasmy of four selected variants reveals stable propagation and clone-specificity. Color bar: allelic heteroplasmy (%). (E) Unsupervised hierarchical clustering of TF1 clones. Color: primary clones as in (C). (F) Between-clone and within-clone accuracy of identifying the most-recent common ancestor (MRCA) per trio of clones based on mtDNA mutational profile. (G) Schematic of mitochondrial relatedness matrix Kmito where each pair of clones is scored based on mitochondrial genotype similarity. (H) Random effects model for variance decomposition of epigenomic peaks. (I) Two examples of peaks inherited in clonal lineages. Peaks represent the sum of open chromatin for the clones with the most samples.

Article Snippet: TF1 Cell Culture TF1 cells (ATCC) were maintained in Roswell Park Memorial Institute Medium (RPMI) 1640, 10% fetal bovine serum (FBS), 2mM L-Glutamine and 2ng/ml recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (Peprotech) and incubated at 37°C and 5% CO2.

Techniques: Clone Assay

Journal: Cell

Article Title: Lineage Tracing In Humans Enabled By Mitochondrial Mutations And Single Cell Genomics

doi: 10.1016/j.cell.2019.01.022

Figure Lengend Snippet: (A) Coverage of mouse mitochondrial genome by six scRNA-seq methods. Shown is the fraction (%) of the mitochondrial genome (y axis) covered by reads from each of six methods (color code), at different levels of coverage (x axis). (B) Agreement in allelic heteroplasmy estimates from single cell whole genome sequencing (WGS) and scRNA-seq from the same single cells. Shown is the allele frequency for scRNA- (y axis) and scWGS-seq (x axis) based estimates for two cell lines (HCC827: orange; SKBR3: purple). Two examples of RNA-specific changes are highlighted. (C-F) Identification of mitochondrial mutations by scRNA-, scATAC- and scMito-seq in three TF1 clones. (C) Bulk and single cell data collected for three TF1 clones (boxed). Each clone (n = 3) was processed with variable numbers of single-cell libraries (k). (D) Agreement in allelic heteroplasmy estimates from bulk ATAC- (x axis) and bulk RNA-seq (y axis) from three indicated TF1 clones (as in (C)). Two examples of RNA-specific changes are highlighted. (E) Coverage of the mitochondrial genome of the TF clone G10 by each indicated assay. Inner circle: mitochondrial genome; middle blue outline: coverage; outer grey circle: genome coordinates. For single cell assays, coverage is the sum of single cells. (F) Four clone-specific mutations that are reliably detected by various single-cell assays with heteroplasmies as low as 3.8%. Each boxplot shows the % heteroplasmy (y axis) of one mutation across scATAC-, scMito- and scRNA-seq in the three TF1 clones (color code as in (C)). Dots: individual cells.

Article Snippet: TF1 Cell Culture TF1 cells (ATCC) were maintained in Roswell Park Memorial Institute Medium (RPMI) 1640, 10% fetal bovine serum (FBS), 2mM L-Glutamine and 2ng/ml recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (Peprotech) and incubated at 37°C and 5% CO2.

Techniques: Sequencing, Clone Assay, RNA Sequencing, Mutagenesis

Journal: Cell

Article Title: Lineage Tracing In Humans Enabled By Mitochondrial Mutations And Single Cell Genomics

doi: 10.1016/j.cell.2019.01.022

Figure Lengend Snippet: (A) Experimental overview. TF1 cells were infected with a lentiviral vector expressing the mNeonGreen gene and a 30bp random barcode in the untranslated region (Figure S3A). 25 cells were sorted and expanded, followed by bulk ATAC-seq and scRNA-seq. (B) Filtering of high confidence mutations. Base quality (BQ) scores from scRNA- (y axis) and from bulk ATAC-seq (x axis). White box: high-confidence variants detected by both technologies (BQ >20) (STAR Methods). (C) Allele frequencies determined by the sum of single cells from scRNA-seq (y axis) and bulk ATAC-seq (x axis). Black – filtered; red – retained. (D-F) mtDNA inferred clones agree with barcode-based clones. (D) Hierarchical clustering of TF1 mitochondrial genotyping profiles (rows) for cells assigned to annotated barcode groups (columns) (from Figure S3A). Color bar: Heteroplasmy (% allele frequency). (E) Cell-cell similarity from mitochondrial mutations called in (C). Column and rows are annotated by barcode group. (F) Between-group accuracy of identifying the most-similar pair per trio of clones based on mtDNA mutational profile using detected barcodes as a true positive.

Article Snippet: TF1 Cell Culture TF1 cells (ATCC) were maintained in Roswell Park Memorial Institute Medium (RPMI) 1640, 10% fetal bovine serum (FBS), 2mM L-Glutamine and 2ng/ml recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (Peprotech) and incubated at 37°C and 5% CO2.

Techniques: Infection, Plasmid Preparation, Expressing, Clone Assay