GSM7232639: CLS058_popD_GEX; Homo sapiens; RNA-Seq
Bone marrow aspirate was obtained under sterile conditions from the iliac crest from each of the 5 healthy adults. Mononuclear cells were isolated by Ficoll density gradient centrifugation and enriched by a custom-designed negative selection EasySep cell isolation kit from StemCell that removes CD66b+/GPA+/CD3+/CD14+ cells to limit sorting time of fragile BM ASC. Enriched mononuclear cells were stained with the following anti-human antibodies: IgD–FITC (Cat. no. 555778; BD Biosciences), CD3-BV711 (Cat. no. 317328; BioLegend), CD14-BV711 (Cat. no. 301838; BioLegend), CD19-PE-Cy7 (Cat. no. 560911; BD Biosciences), CD38-V450 (Cat. no. 561378; BD Biosciences), CD138-APC (Cat. no. 130-117-395; Miltenyi Biotech), CD27-APC-e780 (Cat. no. 5016160; eBiosciences), and LiveDead (L34966; Invitrogen). ASC subsets were FACS-sorted on a BD FACSAria II using a standardized sorting procedure with rainbow calibration particles to ensure consistency of sorts between individuals. ASC subsets were sorted as: popA (CD19+CD38hiCD138-), popB (CD19+CD38hiCD138+) and popD (CD19-CD38hiCD138-) FACS-sorted cells were kept on ice until proceeding with 10x Genomics processing. Cells were centrifuged at 500xg for 10 minutes at 4C to remove media. Cells were then washed twice in 0.04% BSA in PBS. During last wash, media was aspirated and cell volume was measured using a pipette. The whole sample was then taken for processing using the 10x Genomics 5' v1 Single Cell platform. V(D)J and 5' Gene Expression (GEX) libraries were constructed for each sample, following 10x Genomics protocol. QC for each library was performed at each step by Bioanalyzer. Final libraries were quantified by kappa qPCR and sequenced on a NovaSeq.