GSM9465238: Sequencing Lane 1,RNA; Homo sapiens; RNA-Seq
Freshly isolated BMNCs from NSG-SGM3 mice engrafted with CD34⁺ cord blood–derived hematopoietic stem cells, collected from the femurs and tibias, were suspended in Human FcR Blocking Reagent (Miltenyi Biotec, catalog #130-059-901), diluted in Filtered DPBS (Corning, catalog #21-031-CM) + 0.5% BSA (Fisher, catalog #F2442-500ML) and incubated for 10 minutes at 4ºC.  Brilliant Staining Buffer (BD), TotalSeqTM Oligonucleotide anti-Human Hashtag antibodies (Biolegend; C0251 catalog # 394661, C0252 catalog # 394663, C0253 catalog # 394665, C0254 catalog # 394667, or C0258 catalog # 394675), surface antibody mix (including human anti-CD45 BV510, Biolegend) were added to the FcR Blocking solution and incubated at RT for 30 minutes in the dark. Cells were washed with filtered DPBS + 0.5% BSA, filtered using 30 µm filters (Sysmex), and resuspended in filtered DPBS + 2% BSA + DAPI. Cell populations were purified using Fluorescent Activated Cell Sorting (FACS), using the Aria3 Instrument (BD Biosciences). Upon purification and collection of cell populations in Protein Lo-Bind Eppendorf Tubes, samples were pooled together based off cell number  and washed with filtered Cell Staining Buffer (Biolegend, catalog # 420201). Cells were Fc Blocked using TruStain FcX (Biolegend) at 4ºC for 10 minutes and then stained with TotalseqTM Universal Cocktail (Biolegned, catalog #399905) for 30 minutes at 4ºC. The cells were washed once with Cell Staining Buffer and twice with DPBS + 0.04% BSA and then submitted to the MSKCC Integrative Genomics Core for sequencing. Standard library construction was performed using a 10x Chromium Single Cell Immune Profiling Gel Bead Kit.