Cd45 hi30

Phenotypic analysis was performed on NPA cells and PBMCs using Live/Dead Blue; lineage markers CD3 (SK7; BD), CD19 (HIB19; Biolegend), CD20 (L27, BD), CD45 (HI30; BD), CD56 (HCD56, BD) and CD66abce (TET2, Miltenyi Biotec); HLA-DR (TU36, Life Technologies), CD14 (M5E2, BD), CD16 (3GE, Biolegend), CD11c (B-Ly6, BD), CD1c (AD5-8E7; Miltenyi), CD141 (AD5-14H12; Miltenyi), CD123 (7G3; BD); maturation markers CD83 (HB15e, Biolegend) and CD86 (2331; BD); adhesion marker CD62L (SK11, BD); and migration markers CCR2 (K036C2, Biolegend) and CCR7 (150503: BD); and fixed with 1% paraformaldehyde for flow cytometry. Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) and analysed using FlowJo software v10 (Tree Star).

Validation of high confidence candidate mutations was carried out in the original samples as well as in primary, non-expanded MSCs, which were FACS sorted from viably frozen BM specimen of the corresponding patient samples, in whose MSC cultures the mutations were detected. Viable cells were stained and sorted for the parameters Sytox^®−, CD45−, CD235a−, CD31−, CD271+ (CD45-HI30, BD Bioscience, PerCP-Cy 5.5, Cat No: 564106, 1:100; CD235a-GA-R2, BD Bioscience, APC, Cat No 551336, 1:100; CD31-WM59, Biolegend, APC.C7 Cat No 56365, 1:1000; CD271-ME20.4, Biolegend, FITC Cat No 345104, 1:20; Sytox^®, Thermo Fisher) on a BD FACS Melody sorting device. In addition, a second FACS strategy (Sytox−, CD45−, CD235a−, CD31+/−) was employed to enrich non-hematopoietic cells. Cells were directly sorted into Qiagen ALT lysis buffer and the whole genome amplified with the Qiagen repliG Kit in the majority of cases. For validation with targeted deep sequencing, single PCRs surrounding mutational sites (Primers in Supplemental Data 1) were carried out with subsequent library generation using the Nextera XT kit (Illumina). Samples were pooled and sequenced with MiSeq v3 chemistry at a mean coverage of 8699x. Quantification was then derived from bamfiles after bwa mem (v0.7) and picard MarkDuplicatevs (2.20). For further detailed methods, please see the Supplemental methods section.