title: GSM6102905: Cytokine hashtags for samples 27; Mus musculus; RNA-Seq
library_construction_protocol: Single-cell suspensions of freshly collected lymph nodes were generated using enzymatic digestion with Collagenase IV, Dispase, and DnaseI in RPMI. To rebalance cell types, a biotinylated anti-CD3 and anti-CD19 antibody cocktail was incubated with the cells, then streptavidin beads were added and the samples were processed using MACS MS columns according to the manufacturer's protocol. After cell sorting, a small fraction of the CD3+ or CD19+ cells was pooled with CD3-/CD19- cells for optimal representation of all cell types, and proceeded immediately to scRNA-seq. Cell hashing was used to combine multiple samples into the same single-cell emulsion channel. The lymph node cells were stained with TotalSeq antibodies (BioLegend anti-mouse Hashtags), washed 5 times and pooled in ice-cold PBS with 0.04% BSA according to the manufacturer's protocol. Next, 55,000 cells were loaded onto a 10x Genomics Chromium instrument (10x Genomics) according to the manufacturer's instructions. The scRNA-seq libraries were processed using Chromium Single Cell 3' Library & Gel Bead v3 Kit (10x Genomics) with BioLegend's protocol for generating hashtag libraries. Quality control for amplified cDNA libraries and final sequencing libraries was performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). scRNA-seq and hashing libraries were normalized to 4nM concentration and pooled. The pooled libraries were sequenced on the NovaSeq S4 platform for gene expressions, targeting an average sequencing depth of 20,000 reads per cell for gene expression libraries. Hashtag libraries were sequenced on NovaSeq S4 or SP targeting 5,000 reads per cell.