Extended Data Fig. 10: Depletion of NIPBL in primary bone marrow haematopoietic progenitors enriches for polymorphonuclear cells.
From: Nuclear morphology is shaped by loop-extrusion programs
a, NIPBL-FKBP12F36V-EYFP ECOMG progenitors were electroporated with crRNAs that target four different genomic regions in NIPBL, named NIPBL-crRNA1–4, tracrRNAs conjugated with ATTO-550 and Cas9. Electroporated ECOMG progenitors were cultured for three days in the presence of β-oestradiol and examined for CD11b and Ly6G expression. Upper panels show transfection efficiencies as reflected by ATTO-550 expression. ECOMG cells that were not electroporated served as a control. As an additional control ECOMG cells were electroporated with negative crRNPs (control RNPs) lacking specificity for genomic sequences (IDT). b, ECOMG cells electroporated with NIPBL-crRNPs 1 + 2 and control crRNPs were analysed for the expression of CD11b and Ly6G. c, ECOMG cells were electroporated with NIPBL-crRNPs 1 + 2. Ly6G+ cells were sorted after 72 hrs. Sorted samples were examined by DNA sequencing for the presence of indels. d, Lin- primary bone marrow progenitors were electroporated with crRNPs 1 + 2 targeting NIPBL. Electroporated cells were cultured for three days in the presence of SCF and Flt3L and examined for transfection efficiencies and for CD11b and Ly6G expression. e, Three days after electroporation ATTO-550− and ATTO-550+ cells were sorted and analysed for the presence of indels. f, ATTO-550− and ATTO-550+ cells were sorted and analysed for nuclear morphology using Wright–Giemsa staining. Table indicates the percentages of progenitors and differentiated progeny in cells transfected with crRNPs (1 + 2) that target NIPBL. A total of 400–500 cells for each condition was examined. All experiments were performed independently at least three times.
