Antibody coated microbeads

6–8-week-old, female NBSGW (NOD.Cg-KitW-41J Tyr+ Prkdcscid Il2rgtm1Wjl/ThomJ, Stock#026622) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in a conventional facility at ambient temperature of 20-22 °C with 40-60% humidity and with a 12-h day-night light cycle. Frozen CD34⁺ cells isolated from adult human peripheral blood as described above were thawed in X-VIVO medium (Lonza, #04-380Q) and electroporated with CRISPR/Cas9 RNPs as described above. 2 million control or SpCas9 RNP edited CD34⁺ cells were equally administered into 5 mice via tail-vein injection. Mouse bone marrows were harvested 16 weeks post-transplantation to assess engraftment and cell lineage composition using anti-CD45 (BD Biosciences, #560367), human-specific PE anti-CD49d (BioLegend, # 304304), PE/Cy7 anti-CD71 (BioLegend, #334112), FITC anti-human CD235a (BioLegend, #349104), PE anti-CD33 (BioLegend, #366608), APC anti-CD19 (BioLegend, #302212) and anti-Fetal-Hemoglobin (HbF-1), and anti-APC (Thermo Fisher Scientific, #MHFH05) antibodies. CD235a⁺ erythroblasts were purified using antibody-coated microbeads (Miltenyi Biotec, #130-050-501) and analyzed for indels, intracellular HbF staining, hemoglobin HPLC, and RNA analysis.

Adipose tissue from C57BL/6 mice was minced and digested with collagenase D (Roche) in a shaking water bath (37C, 225rpm, 40min). The digest was centrifuged at 400g for 10 min. Pelleted stromal vascular cells were filtered (40μm) and then washed with PBS and subjected to additional negative selection (CD31⁻/lineage⁻) adapted from previously performed methods^{44 (link)} using antibody coated microbeads (Miltenyi Biotec). Cells were cultured to confluence in collagen-coated plates and stimulated with dexamethasone, insulin and 3-isobutyl-1-methylxanthine to induce adipogenic differentiation. For genetic loss of function assays, validated shRNA sequences (Broad, Ube2e2: TRCN0000040962; Atxn1: TRCN0000240655) or scramble sequence were subcloned into a retroviral vector (pMKO.1). Gene knock-down efficiency was confirmed by qPCR in 3T3L1 cells, in each instance reproducibly achieving a minimum of 60% reduction of transcriptional activity. Differentiation into mature lipid-containing adipocytes was determined by oil-red-o (ORO) staining and quantified by measuring alcohol-extracted ORO dye at optical density 520 nm (OD₅₂₀).