powerplex 16hs multiplex str system  (Promega)

Journal: Nature Communications

Article Title: Synthetic modeling reveals HOXB genes are critical for the initiation and maintenance of human leukemia

doi: 10.1038/s41467-019-10510-8

Figure Lengend Snippet: NLTB-transduced CB cells do not develop into clonally rearranged leukemias in vitro. a , b BIOMED-2 TCRG clonality assay. a CB cells were transduced with N + LTB lentiviruses and cultured on OP9-DL1 feeders for up to 28 days. Doubly transduced (G+C+) and nontransduced (G−C−) subsets of cells were FACS sorted and genomic DNA extracted for analysis. No dominant peaks indicative of clonal T-cell populations were identified in any of these samples. b Doubly transduced (G+C+) CB cells were FACS sorted from day 24 in vitro cultures, as well as from the leukemic mice into which the cultured cells were injected. Genomic DNA was then extracted, and the distribution of amplified TCRG DNA fragments analyzed by GeneScan (Applied Biosystems). Dotted red lines are overlaid to facilitate comparison of peak sizes between samples. Peak sizes are reproducible within less than 0.5 bp. c STR profiling of CB leukemias. Genomic DNAs from primary CB leukemias were profiled by Promega PowerPlex 16HS assay. STR patterns from four different individual donors (A, B1, C, D) can be discerned. The minor B2 pattern was not represented after serial transplant. d ImmunoSEQ TCRG clonality assay. CB cells were transduced with N + LTB lentiviruses and cultured on OP9-DL1 feeders. Culture samples were taken at 14, 28, and 38 days. Doubly transduced (G+C+) and nontransduced (G−C−) subsets were FACS sorted and genomic DNA extracted for ImmunoSEQ TCRG (Survey) analysis by Adaptive Biotechnologies. Stacked reads are plotted for the 25 most frequent rearrangements with each alternating color indicating a unique, clonally rearranged CDR3 DNA fragment

Article Snippet: Cell line authentication by PowerPlex 16HS multiplex STR DNA profiling (Promega) was performed by Genetica DNA Laboratories (Burlington, NC).

Techniques: In Vitro, Transduction, Cell Culture, Injection, Amplification, Comparison

Journal: Nature Communications

Article Title: Synthetic modeling reveals HOXB genes are critical for the initiation and maintenance of human leukemia

doi: 10.1038/s41467-019-10510-8

Figure Lengend Snippet: Synthetic CB leukemias exhibit clonal evolution in vivo and are highly similar to PDXs. a Venn diagram of single nucleotide variants (SNVs). FACS-sorted G+C+ cells from bone marrow or spleen of a low-level engrafted primary recipient mouse were injected into secondary recipients. G+C+ cells were FACS sorted from clinically morbid secondary recipients and genomic DNA extracted for whole exome sequencing. b Unsupervised hierarchical clustering of synthetic NLTB CB leukemias and PDX T-ALLs based on RNA-seq data. Following batch correction with ComBat, correlation distances (1 − Spearman coefficient) across 39 samples (17 synthetic CB leukemias + 22 PDX) were calculated using the top 1000 variable genes within the PDX sample set. Individual CB donors were discriminated by STR profiling. Darker blue indicates greater similarity. PDX RNA-seq data are from the PRoXe repository (NCBI SRA Accession SRP103099). Color scale indicates (1 − Spearman) correlation distance. Darker blue indicates greater similarity. c TCR clonality by MiXCR analysis of RNA-seq data. Distinct CDR3 regions are indicated by alternating colors within each sample; matching colors across samples are coincidental and do not indicate sequence identity. Mature T-cell control shows polyclonal TRA/TRB. The depicted G+C+ CB leukemias show mono/oligoclonal patterns for TRG/TRD and TRB, respectively. The depicted G+C− CB leukemia shows oligoclonal TRB

Article Snippet: Cell line authentication by PowerPlex 16HS multiplex STR DNA profiling (Promega) was performed by Genetica DNA Laboratories (Burlington, NC).

Techniques: In Vivo, Injection, Sequencing, RNA Sequencing, Control