GSM7780534
20,000 LSK cells were sorted from a pool of four 2-months-old C57Bl/6J mice. The CITE-Seq protocol described in Muench et al. was applied to a LSK cells using a collection of 65 TotalSeq antibodies. For staining optimization, individual antibodies were titrated on ckit+ bone marrow cells by first conjugating the TotalSeq-A antibodies to an oligo-dT conjugated to Alexa Fluor 647 at a 1:1.5 molar ratio, before staining the cells at different dilutions. The prepared samples were washed and run on a BD LSRFortessa flow cytometer to determine optimal concentrations. For FACS-sorting total BM cells were collected from 3 C57/Bl6 male mice and enriched for ckit+ cells (Miltenyi). Cells were stained with fluorochrome-conjugated antibodies in staining buffer (1% FBS, PBS) for 30 minutes at 4C and washed three times in staining buffer at 4C. Cells were FACS-sorted and resuspended in 50uL of TotalSeq-A antibody cocktail for 30min at 4C and washed one time. Cells were resuspended at 1,000 cells/L, and 16,000 cells were loaded in 1 port of 10X Genomics Chromium Controller Instrument. Genomics single-cell 3' v3 assay protocol was performed according to manufacturer's recommendation until the cDNA amplification step. The antibody derived tags (ADTs) were processed according to the CITE-Seq protocol detailed by BioLegend (Biolegend Inc, https://www.biolegend.com/en-us/protocols/totalseq-a-antibodies-and-cell-hashing-with-10x-single-cell-3-reagent-kit-v3-3-1-protocol). The GEX library was generated following regular protocol of 10X Genomics. The final GEX library was sequenced at 50,000 reads per cell with paired-end 150 on Illumina NovaSeq platform (Novogene). The final ADT library was sequenced at 10,000 reads per cell with paired-end 50 on Illumina NovaSeq platform (Novogene).