scRNA-Seq of Homo sapiens: osteoclast progenitors
Whole transcriptome analyses at single cell level and profiling of oligoconjugated antibodies were performed on FACS-sorted GFP+ alive cells using the BD Rhapsody Single-Cell Analysis System (BD, Biosciences). Specifically, human CD14+CD16- monocytes were isolated from PBMCs and transduced with lentivectors encoding the active NOTCH1-E isoform or empty vector as control. Transduced GFP+ cells were in vitro RANKL stimulated and expanded for 5 days and subsequently purified by FACS-sorting as previously reported (Dworzak MN et al., Leukemia, 2004). Isolated cells from each sample were labeled with the BD Single-Cell Multiplexing Kit (cat. 633781, BD Biosciences) using sample tags #04 and #06 for empty vector and Notch1-transduced cells respectively, as well as 31 BD AbSeq Ab-Oligos for 1 hour, following the manufacturers protocol. The cell viability and concentration were determined with the BD Rhapsody Scanner system after staining with viability dyes, Calcein AM (1:200 dilution; cat. #C1430, ThermoFisher) and DRAQ7TM (1:200 dilution; cat. #564904, BD Biosciences), and incubation for 5 min at 37C. Cells were counted using the Improved Neubauer Hemocytometer (INCYTO). Afterward, GFP+ cells for each sample were pooled equally in 650ml cold BD Sample Buffer and a BD Rhapsody cartridge was loaded with 10,000 pooled cells for single cell separation. Single cells were isolated using Single-Cell Capture and cDNA Synthesis with the BD Rhapsody Express Single-Cell Analysis System according to the manufacturers recommendations (BD Biosciences). Based on the number of viable cells revealed and captured on the beads, the final resuspension volume was calculated to subsample and sequence about 2,000 cells. Whole transcriptome, Sample Tag, and BDTM AbSeq amplification was performed with the BD Rhapsody Whole Transcriptome and AbSeq Amplification Kit (cat. #633774), following the manufacturers instructions. Unwanted PCR products and other small molecules were excluded performing a side cleanup using the AMPure XP Beckman magnetic beads (cat. #A63880, Beckman Coulter). DNA quantity and quality control were performed using the QubitTM dsDNA HS Assay Kit (cat. # Q32851, ThermoFisher Scientific) and the electrophoresis system Agilent 2200 TapeStation, cartridge (cat. #5067-5584). Sequencing was performed in paired-end mode (2*75 cycles) on NextSeq 500 System (Illumina) with the NextSeq 500/550 High Output Kit v2.5 (150 Cycles) chemistry to reach a depth of 75,000 reads for WTA, 21,500 reads for AbSeq and 500 reads for SMK per cell for a total of 97,000 reads per cell on average.