Journal: Nature methods

Article Title: 3D mapping and accelerated super-resolution imaging of the human genome using in situ sequencing.

doi: 10.1038/s41592-020-0890-0

Figure Lengend Snippet: Fig. 3 | Every-pixel analysis pipeline on 36plex-5K. a, Sequencing rounds (step 1) were analyzed at the level of individual pixels using tier 1 parameters with thresholds for signal intensity and pixel patch size (step 2), and pixel patches were then decoded (step 3). Missing targets and FPs were filtered by reanalyzing images with tier 2 parameters (step 4) to produce traces (step 5). Tier 2 decreased the thresholds for signal intensity and pixel patch size, subsampled barcodes and applied filters for chromosome territories. Barcodes and color codes were designated as follows: 1, FITC; 2, Cy3; 3, TxRed; 4, Cy5. b, Tier 2 detection efficiency of 36plex-5K after sequencing off both streets; 80.2% ± 7.3% of targeted regions were detected in 611 cells from 15 replicates. c, Detection efficiencies from individual replicates are shown, with chromosomal targets on the x axis. The dashed red line marks the mean of all chromosomal targets. 3qR3 and 5pR3 shared a barcode and were not included. Error bars represent the 95% bootstrap confidence interval (CI) of the mean. c, Chromosome traces of Fig. 2c nucleus after tier 2. In total, 64 of 66 (97%) targeted regions were detected; n = 1. d, Ball-and-stick traces of the nucleus referred to in c. Colored spheres represent targets; black spheres represent undetected targets and were positioned by calculating the median proportionate distance between flanking detected spheres. Gray lines between signals denote extrapolations. The asterisks mark the beginning of chromosomes. e, Single-cell pairwise spatial distance matrix after tier 2 detection of the nucleus referred to in b. Homologs are displayed separately. Centroids of targets were used for this and all subsequent spatial distance matrices. Gray lines denote undetected targets. f, 36plex-5K population pairwise spatial distance measurements after tier 1 detection (n = 611 cells from 15 replicates). Homologous target measurements were combined.

Article Snippet: Anti-GAPDH (Abcam: ab9483) used at 1:200 Anti-TOMM20 (Abcam: ab78547) used at 1:500 Anti-macroH2A.1 (Abcam: ab183041) used at 1:250 Donkey Anti Mouse Cy5 (Jackson ImmunoResearch Laboratories: 715-175-150) used at 1:500 from 1.25mg/mL stock Donkey Anti-Rabbit Cy3 (Jackson ImmunoResearch Laboratories: 711-165-152) used at 1:500 from 1.25mg/mL stock Bovine Anti-Goat Alexa Fluor 594 (Jackson ImmunoResearch Laboratories: 805-585-180) used at 1:500 from 1.25mg/mL stock Validation Anti-Alpha Tubulin has been validated by Sigma-Aldrich to be specific in human cell lines (osteosarcoma and breast cancer) using western blotting and in HeLa cells by immunofluorescence microscopy (https://www.sigmaaldrich.com/catalog/product/sigma/ t9026?lang=en®ion=US).

Techniques: Sequencing

Journal: Nature methods

Article Title: 3D mapping and accelerated super-resolution imaging of the human genome using in situ sequencing.

doi: 10.1038/s41592-020-0890-0

Figure Lengend Snippet: Fig. 3 | Every-pixel analysis pipeline on 36plex-5K. a, Sequencing rounds (step 1) were analyzed at the level of individual pixels using tier 1 parameters with thresholds for signal intensity and pixel patch size (step 2), and pixel patches were then decoded (step 3). Missing targets and FPs were filtered by reanalyzing images with tier 2 parameters (step 4) to produce traces (step 5). Tier 2 decreased the thresholds for signal intensity and pixel patch size, subsampled barcodes and applied filters for chromosome territories. Barcodes and color codes were designated as follows: 1, FITC; 2, Cy3; 3, TxRed; 4, Cy5. b, Tier 2 detection efficiency of 36plex-5K after sequencing off both streets; 80.2% ± 7.3% of targeted regions were detected in 611 cells from 15 replicates. c, Detection efficiencies from individual replicates are shown, with chromosomal targets on the x axis. The dashed red line marks the mean of all chromosomal targets. 3qR3 and 5pR3 shared a barcode and were not included. Error bars represent the 95% bootstrap confidence interval (CI) of the mean. c, Chromosome traces of Fig. 2c nucleus after tier 2. In total, 64 of 66 (97%) targeted regions were detected; n = 1. d, Ball-and-stick traces of the nucleus referred to in c. Colored spheres represent targets; black spheres represent undetected targets and were positioned by calculating the median proportionate distance between flanking detected spheres. Gray lines between signals denote extrapolations. The asterisks mark the beginning of chromosomes. e, Single-cell pairwise spatial distance matrix after tier 2 detection of the nucleus referred to in b. Homologs are displayed separately. Centroids of targets were used for this and all subsequent spatial distance matrices. Gray lines denote undetected targets. f, 36plex-5K population pairwise spatial distance measurements after tier 1 detection (n = 611 cells from 15 replicates). Homologous target measurements were combined.

Article Snippet: Anti-GAPDH (Abcam: ab9483) used at 1:200 Anti-TOMM20 (Abcam: ab78547) used at 1:500 Anti-macroH2A.1 (Abcam: ab183041) used at 1:250 Donkey Anti Mouse Cy5 (Jackson ImmunoResearch Laboratories: 715-175-150) used at 1:500 from 1.25mg/mL stock Donkey Anti-Rabbit Cy3 (Jackson ImmunoResearch Laboratories: 711-165-152) used at 1:500 from 1.25mg/mL stock Bovine Anti-Goat Alexa Fluor 594 (Jackson ImmunoResearch Laboratories: 805-585-180) used at 1:500 from 1.25mg/mL stock Validation Anti-Alpha Tubulin has been validated by Sigma-Aldrich to be specific in human cell lines (osteosarcoma and breast cancer) using western blotting and in HeLa cells by immunofluorescence microscopy (https://www.sigmaaldrich.com/catalog/product/sigma/ t9026?lang=en®ion=US).

Techniques: Sequencing