Computational protocol: A Human IPS Model Implicates Embryonic B-Myeloid Fate Restriction as Developmental Susceptibility to B Acute Lymphoblastic Leukemia-Associated ETV6-RUNX1

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Protocol publication

[…] Single cell suspensions were blocked with Fc receptor binding myeloma antibodies and then stained with specific monoclonal antibodies listed in (human primary samples and IPS samples). Before analysis and cell sorting the sample was dissolved in viability dye (). For cell sorting samples were sometimes enriched with CD34 isolation beads (Miltenyibiotec) before staining was performed.Progenitors were identified according to the following surface markers; (LIN-)CD34+CD45RA-: (LIN-)CD19-CD34+(CD38-)CD45RA-(KIT+), IL7R+(KIT+) progenitor: (LIN-)CD19-CD34+(CD38+)CD45RA+IL7R+(KIT+); ProB; (LIN-)CD34+CD19+; PreB; CD34-CD19+ or CD34-CD19+IGM-; CD34+: (LIN-)CD19-CD34+. Some markers were not used on IPS cells; KIT, CD38 and the lineage markers (LIN); CD3, CD8a, CD235a, CD56; CD38 surface expression was affected by culture conditions, as previously reported (), and surface KIT was affected by the addition of exogenous KIT ligand. Samples were analyzed or sorted on an LSR II, FortessaX20, BD ARIA IIu or BD FACSAria III (all from BD Biosciences) and analysis were performed using the FlowJo software. [...] Raw reads were initially processed with PRINSEQ-lite v0.20.4 () to trim low-quality reads. They were further aligned with TopHat2 v2.0.11 () on the GRCh37 human assembly and FPKM values for protein-coding genes were calculated with Cufflinks v2.2.0 () using uniquely mapping reads only. Genes with an FPKM < 5 in every sample were filtered out. The remaining ones were log-transformed using the formula log2 (FPKM+1). Finally, expression values were normalised upon quantiles () prior to the PCA analysis. When building the PCA map based on the expression in primary samples only (FL CS17, FL CS21-22 and Adult BM), the FL CS17 IL7R+ Progenitor sample was detected as a clear outlier. A new map based on all other primary cells was used instead and the FL CS17 IL7R+ Progenitor sample was projected on the new map. When building the PCA map based on the expression of all wild-type samples (hPSC D31 and the aforementioned primary samples), the FL CS17 IL7R+ Progenitor sample rested on the correct region of the PCA map (E). Gene Set Enrichment Analysis (GSEA) was performed with permutation type=gene set and the gene sets used were obtained from Laurenti et al. (, , ). Ellipses on the PCAs were calculated using ggplot stat_ellipse (http://ggplot2.tidyverse.org/reference/stat_ellipse.html) assuming a multivariate t-distribution and a confidence level of 0.9. […]

Pipeline specifications

Software tools FlowJo, PRINSEQ, TopHat, Cufflinks, GSEA, Ggplot2
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens, Caenorhabditis elegans
Diseases Leukemia, Precursor Cell Lymphoblastic Leukemia-Lymphoma