Eep sequencing to targeted exons as previously described.15 Briefly, we analyzed for possible mutations of SETBP1 as well as other genes which have been concomitantly mutated inside the cases with SETBP1 mutation (U2AF1, DNMT3A, NRAS, ASXL1, SRSF2, CBL, IDH1/2, SRSF2, TET2, PTPN11, RUNX1). Each targeted exon was amplified with NotI linker attached to every primer. Immediately after digestion with NotI, the amplicons have been ligated with T4 DNA ligase and sonicated into up to 200bp fragments on average applying Covaris. The sequencing libraries were generated based on an Illumina pair-end library protocol and subjected to deep sequencing on Illumina GAIIx or HiSeq 2000 sequencers in accordance with the regular protocol. Sanger sequencing and allele-specific PCR Exons of chosen genes have been amplified and underwent direct genomic sequencing by typical procedures around the ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA) as previously described.413 Coding and sequenced exons are shown in Supplementary Table eight. All mutations have been detected by bidirectional sequencing and scored as pathogenic if not present in non-clonal paired CD3-derived DNA. When D1 Receptor Inhibitor custom synthesis marginal volume of mutant clone size was not confirmed by Sanger sequencing, cloning and sequencing person colonies (TOPO TA cloning, Invitrogen, Carlsbad, CA) was performed for validations. The allelic presence of p.Asp868Asn and p.Gly870Ser alterations was determined by allelespecific PCR. Primers for SETBP1 sequencing and SETBP1 allele-specific PCR were offered in Supplementary Table 14.Nat Genet. Author manuscript; readily available in PMC 2014 February 01.Makishima et al.PageQuantitative RT-PCR by TaqMan probesAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptTotal RNA was extracted from bone marrow mononuclear cells and cell lines. cDNA was synthesized from 500 ng total RNA using the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). Quantitative gene expression levels have been detected applying cIAP-1 Antagonist Compound real-time PCR with all the ABI PRISM 7500 Quick Sequence Detection Technique and FAM dye labeled TaqMan MGB probes (Applied Biosystems). TaqMan probes for all genes analyzed had been purchased from Applied Biosystems gene expression assays solutions (SETBP1: Hs00210209_m1; HOXA9: Hs00365956_m1; HOXA10: Hs00172012_m1; GAPDH: Hs99999905_m1). The expression amount of target genes was normalized to the GAPDH mRNA. Retrovirus generation pMYs-Setbp1 retrovirus expressing 3xFLAG-tagged wild-type Setbp1 protein and GFP marker was described previously.31 Point mutations of Setbp1 (p.Asp868Asn and p.Ile871Thr) have been generated working with the same construct and QuickChange II site-directed mutagenesis kit (Agilent). Virus was created by transient transfection of Plat-E cells applying Fugene six (Roche). Viral titers have been calculated by infecting NIH-3T3 cells with serially diluted viral stock and counting GFP optimistic colonies 48 hours following infection. Immortalization of myeloid progenitors Immortalization of myeloid progenitors was performed as described.31 Briefly, entire bone marrow cells harvested from young C57BL/6 mice had been first cultured in StemSpan medium (Stemcell Technologies) with ten ng/ml mouse SCF, 20 ng/ml mouse TPO, 20 ng/ml mouse IGF-2 (all from R D Systems), and 10 ng/ml human FGF-1 (Invitrogen) for six days to expand primitive stem and progenitor cells. Myeloid differentiation was subsequently induced by increasing the expanded cells in IMDM plus 20 heat-inactivated horse serum with 100 ng/ml of mouse SCF (PeproTech, Rocky Hill, NJ).