GSM6631695: Lymph Node 1 Protein; Mus musculus; RNA-Seq
Five groups of mice (n=4) were immunized as described before without TLR adjuvant or with individual different TLR adjuvants: Poly:IC, LPS, CpG, R848 in 40 μl sterile PBS 24 hours before harvest. The LLNs were harvested and single cell suspensions were prepared. Cells were stained with flow cytometry antibodies and different TotalSeq A antibodies (BioLegend) according to the different TLR adjuvant treatment. After the staining, single cells suspensions were washed 3 times by resuspension in 4.5 ml HBSS plus 0.5% BSA, followed by centrifugation at 300g for 5 min at 4°C. After the final wash, cells from different treatment groups were resuspended and mixed. Myeloid cells were sorted as shown in Supplementary Figure 1A with the exclusion of Ly6G neutrophils. Sorted cells were centrifugated at 300g for 5 min at 4°C. Supernatants were aspirated, and pellets were resuspended with HBSS plus 0.5% BSA at an approximate concentration of 2.5 × 105 cells/ml. Cell quality and viability were assessed with a Cellometer K2 (Nexcelom Bioscience). All samples had viability >35%. Single cells were then processed using the Chromium Next GEM Single Cell 3' Platform (10X Genomics). Approximately 60,000 cells were loaded on each channel with an average recovery rate of 27,000 cells. Libraries were sequenced on NextSeq2000 (Illumina) with an average sequencing depth of 40,000 reads/cell.