Journal: Experimental Hematology

Article Title: Determination of complex subclonal structures of hematological malignancies by multiplexed genotyping of blood progenitor colonies

doi: 10.1016/j.exphem.2017.09.011

Figure Lengend Snippet: Multiplexed SNP genotyping is an efficient method for genotyping BFU-E colonies . Number of colonies for which a genotype could be called out of 96 colonies for each mutation is shown. Five genes were genotyped by capillary sequencing and multiplexed SNP genotyping. Shaded regions highlight the number of colonies for which the same genotype was called by both technologies.

Article Snippet: The diluted amplified product was loaded onto the 192.24 Dynamic Array IFC for SNP Genotyping (BMK-M-192.24GT, Fluidigm) alongside a sample premixture including ROX reference dye and real-time master mixture.

Techniques: Mutagenesis, Sequencing

Journal: Nature

Article Title: Single cell analysis reveals T cell infiltration in old neurogenic niches

doi: 10.1038/s41586-019-1362-5

Figure Lengend Snippet: a) CD8+ T cells from 6 old (24-29 months) male mice were FACS-isolated from the blood and perfused subventricular zones (SVZs) and analyzed by single cell RNA-sequencing using Smart-seq v4 (4 mice) or nested PCR (2 mice). Left: t-SNE plot of 247 CD8+ T cell transcriptomes, colored by source (blood=red and SVZ=orange). Each dot represents a single T cell transcriptome (n=4 old mice, 25-29 months); b) Expression of Ifng (IFNγ) and the checkpoint gene Pdcd1 (PD-1), represented as log-normalized counts. Each dot represents expression levels in one single cell. n=247 cells from 4 old (25-29 months) mice. Ifng *** FDR =2.89×10 −12 , Pdcd1 *** FDR =1.29×10 −40 , MAST differential expression test; c, d) Clonality of T cells in the blood or SVZs of old mice determined by TCR sequencing from Smart-seq v4 data using TraCeR (Mouse 1-4) (d) or by nested PCR of the TCR transcripts (Mouse 5, 6) (e). TCR-β sequence clones are ordered by decreasing frequency. T cell source is indicated in the bottom row. Dashed lines indicate transitions from expanded to unique TCR-β sequence clones; e) Percentage of T cells isolated from the SVZ or blood found in clones of increasing sizes; f) Venn diagram showing lack of overlap between T cell clones from old blood and brain (SVZ) for Mouse 1-4. Clones were designated as clonally expanded if the same TCR β-chain sequence was detected in multiple T cells.

Article Snippet: Reverse transcription was performed directly on the chip using the SMARTer chemistry from Clontech, and PCR was also performed on the chip using the Advantage PCR kit (SMARTer Ultra Low RNA Kit for the Fluidigm C1, Clontech #634832).

Techniques: Isolation, RNA Sequencing, Nested PCR, Expressing, Quantitative Proteomics, Sequencing, Clone Assay

Journal: Nature

Article Title: Single cell analysis reveals T cell infiltration in old neurogenic niches

doi: 10.1038/s41586-019-1362-5

Figure Lengend Snippet: a) t-SNE projections of 247 CD8+ T cell transcriptomes from old blood and old SVZ (as in ), colored by experimental replicate and individual mouse; b) Expression of the top 50 differentially expressed genes upregulated for 247 CD8+ T cells isolated from old blood or old brain (SVZ) of old mice. Heat map of log-normalized counts, with single cells clustered by the expression of the genes shown in the plot. c) Expression of Ifng in T cells from blood or brains (SVZs) of 2 old (24 months) mice, as measured by nested PCR. Mean +/− s.e.m. of percent of T cells positive for Ifng . Nested PCR does not provide a quantitative metric but rather a binary determination of whether the T cell expresses the transcript for the cytokine or not. Percent of cells isolated from the SVZ or blood expressing Ifng is shown; d, e) Clonality of T cells isolated from the blood and perfused brain of old mice represented with the same X axis to enable direct comparison of clone sizes from different mice (the data are the same as , ). TCR sequences were extracted from single cell RNA-sequencing data using TraCer (Old Mouse 1, 2, 3, and 4) (d) or by nested PCR of the TCR transcripts (Old Mouse 5 and 6) (e). For each mouse TCR-β sequence clones are ordered from left to right in order of decreasing frequency in the top row. The source of the T cell is indicated in the bottom row. f) Venn diagram showing lack of overlap between T cell clones from separate mice, for the four mice for which their TCR repertoire was analyzed by single cell RNA-seq via TraCeR. TCR-β sequences were used, and all unique sequences were only counted once; g, h) Venn diagram showing lack of overlap between T cell clones from the old blood and old SVZ (g) or separate mice (h), for the two mice whose TCR repertoire was analyzed by nested PCR. TCR-β sequences were used, and all unique sequences were only counted once.

Article Snippet: Reverse transcription was performed directly on the chip using the SMARTer chemistry from Clontech, and PCR was also performed on the chip using the Advantage PCR kit (SMARTer Ultra Low RNA Kit for the Fluidigm C1, Clontech #634832).

Techniques: Expressing, Isolation, Nested PCR, Comparison, RNA Sequencing, Sequencing, Clone Assay