GSM6598109: sample676+sample681 blood HTO; Mus musculus; RNA-Seq
Cells were incubated for 10 min with Fc block to block nonspecific antibody binding. Subsequently, cells were incubated with a pool of 190 barcoded TotalSeq™-A antibodies (Biolegend) for 30 min at 4 °C mixed with an antibody against CD45 (clone 30-F11, Thermo Fisher Scientific). After labelling, cells were washed with FACS buffer and CD45+ immune cells were sorted with BD FACS Aria™ II cell sorter (BD Biosciences). Cells were sorted, spun down, and resuspended in PBS with 0.04% BSA at an estimated final concentration of 1,000 cells/μL. Cellular suspensions (target recovery of 10,000 cells) were loaded on a GemCode Single-Cell Instrument (10x Genomics, Inc., Pleasanton, CA, USA) to generate single-cell Gel Bead-in-EMulsion (GEMs). Single-cell RNA-sequencing libraries were prepared using GemCode Single-Cell 3ʹ V3.1 Gel Bead and Library Kit (10x Genomics) according to the manufacturer's instructions.  The cDNA amplification mix was modified to include ADT PCR additive primer (5'-CCTTGGCACCCGAGAATT*C*C-3' where * indicates a phosphorotioate bond) and HTO PCR additive primer (5'-GTGACTGGAGTTCAGACGTGTGC*T*C-3'). Size selection via a SPRIselect Reagent kit (Beckman Coulter, Brea, CA, USA) was used to separate the amplified cDNA molecules for 3′ gene expression and cell surface protein library construction. Sequencing libraries were loaded on an Illumina HiSeq4000 or an Illumina NovaSeq 6000 flow cell with sequencing settings according to the recommendations of 10x Genomics Inc. (read 1: 26 cycles; read 2: 98 cycles; index i7: eight cycles; index i5: no cycles, pooled in a 75:20:5 ratio with a 115 pM loading concentration + 1% PhiX for the combined 3′ gene expression and cell surface / hashing libraries respectively).