Illumina NovaSeq 6000 paired end sequencing; Whole exome sequencing of CD14+ CMML patient cells or of iPS cells derived from CMML patient cells
Whole exome sequencing of CD14+ CMML patient cells or of iPS cells derived from CMML patient cells
Peripheral blood was collected on ethylenediaminetetraacetic acid from patients with a CMML diagnosis according to the World Health Organisation criteria. Peripheral blood mononucleated cells were separated on Fycoll-Hypaque. Peripheral blood CD14+ monocytes were sorted with magnetic beads and the AutoMacs system (Miltenyi Biotech, Bergish Gladbach, Germany). Control samples were peripheral blood CD3-positive T lymphocytes sorted with the AutoMacs system. iPS-derived cells were generated using standard protocol from CD34+ cells. Briefly, CD34+ cells were infected with non-integrated Sendai virus encoding the human reprogramming factors Klf4, Oct4, Sox2, and c-Myc. Two days later, cells were plated on murine embryonic fibroblast (MEF) inactivated by gamma irradiation (RnDSystems, Lille, France), then transferred on day 7 in iPSC medium containing DMEM/F12, GlutaMAX supplement (ThermoFisher Scientific). Medium was replaced every other day for 3-4 weeks before manually picking colonies with human iPSC morphology and expanding them. DNA was extracted from cell samples using Qiagen commercial kits according to manufacturer recommendation. 200ng of genomic DNA was sheared with the Covaris S2 system (LGC Genomics). DNA fragments were end-repaired, extended with an 'A' base on the 3′-end, ligated with paired-end adaptors and amplified (10 cycles) using a Bravo automated platform (Agilent technologies). Exome-containing adaptor-ligated libraries were hybridized for 24 h with biotinylated oligo RNA baits, and enriched with streptavidin-conjugated magnetic beads using SureSelect (Agilent technologies). The final libraries were indexed by PCR
Experimental Factor: cell type
lymphocyte
Experimental Factor: phenotype
CD3 positive