GSM7232651: CLS055_popD_VDJ; Homo sapiens; OTHER
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-) Cells were counted using a Bio-Rad TC10 cell counter and verified via hemocytometer. Cell numbers were adjusted to 1,000 cells per μl to allow for 10,000 single cells per sample loaded in the 10x Genomics Chromium device. The manufacturer's standard protocol for Chromium Next GEM Single Cell 5' Reagent Kit, v2 and Chromium Single Cell Human BCR Amplification kit was used to generate libraries. Libraries were sequenced on an Illumina NovaSeq (paired-end; 2 × 150 bp; read 1:26 cycles; i7 index: 8 cycles, i5 index: 0 cycles; read 2: 98 cycles) such that more than 70% saturation could be achieved with a sequence depth of 5,000 reads per cell for VDJ libraries