GSM7123801: Spleen, WT, Rep2, ADT-derived cDNA; Mus musculus; OTHER
DCs from two WT mice were enriched by magnisort negative selection and 4 million cells were stained with FACS antibodies, TruStain FcX Block (BioLegend, 101320) and the mouse cell surface protein antibody panel containing 160 oligo-conjugated antibodies (TotalSeq-A, BioLegend) and 9 TotalSeq-A isotype controls. 50.000 to 60.000 cDC1s (CD64negCD11cposMHCIIposXCR1posCD172aneg), 10.000 cDC2s (CD64negCD11cposMHCIIposXCR1negCD172apos) and 10,000 random live cells were FACS sorted and loaded into a Chromium Controller (10X Genomics). Sorted single-cell suspensions were resuspended at an estimated final concentration of 1000 cells/µl and loaded on a Chromium GemCode Single Cell Instrument (10x Genomics) to generate single-cell gel beads-in-emulsion (GEM). The scRNA-Seq libraries were prepared using the GemCode Single Cell 3' Gel Bead and Library kit, version NextGEM 3.1 (10x Genomics) according to the manufacturer's instructions with the addition of amplification primer (3 nM, 5'CCTTGGCACCCGAGAATT*C*C) during cDNA amplification to enrich the TotalSeq-A cell surface protein oligos. Size selection with SPRIselect Reagent Kit (Beckman Coulter) was used to separate amplified cDNA molecules for 3' gene expression and cell surface protein construction. TotalSeq-A protein library construction including sample index PCR using Illumina's Truseq Small RNA primer sets and SPRIselect size selection was performed according to the manufacturer's instructions. The cDNA content of pre-fragmentation and post-sample index PCR samples was analyzed using the 2100 BioAnalyzer (Agilent). Sequencing libraries were loaded on an Illumina NovaSeq flow cell at VIB Nucleomics core with sequencing settings according to the recommendations of 10x Genomics, pooled in an 85:15 ratio for the combined 3' gene expression and cell surface protein samples, respectively.