EDL Migratory SIRPa+ DC2 WT/S6KO BD Rhapsody Single Cell (Lane 1)
A total of 26,000 SIRPa+ DC2s per genotype were sorted from the pooled ear draining lymph nodes of 3 C57BL/6 or 3 STAT6-KO mice. Sorted cells were collected into 150 L of BD Stain Buffer (BD Biosciences) supplemented with 5% FCS. Cells were labelled with sample tags from the Mouse Immune Single-Cell Multiplexing Kit (anti-mouse CD45, Clone 30-F11, BD Biosciences) following the manufacturers instructions, washed, resuspended in BD Sample Buffer (BD Biosciences), counted, combined at 5,000 cells per genotype. A BD Rhapsody nanowell cartridge was primed and loaded with 10,000 hashtagged cells (BD Mouse Immune Single-Cell Multiplexing kit) followed by Capture Beads (BD Biosciences). After washing and cell lysis, the cell capture beads were retrieved for reverse transcription as per manufacturers protocol (BD Biosciences, Single Cell Capture and cDNA Synthesis). cDNA Libraries were prepared using mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library kits and Protocol (BD Biosciences, mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library kits according to the manufacturer's instructions. The quality of the final libraries was assessed by using a TapeStation 4150 (Agilent) with High Sensitivity D5000 ScreenTape and quantified using a Quantus Fluorometer (Promega). WTA and Sample Tag library were diluted to 4nM and mixed together at a ratio of 99.63% WTA library to 0.37% SMK library. The libraries were loaded on an S1 flow cell (2x75 cycle) and paired-end sequenced at >200,000 reads/cell depth on a Novaseq 6000 Sequencer (Illumina) at the Kinghorn Centre for Cellular Genomics, Garvan Institute, Sydney, Australia. 20% PhiX was added to the sequencing run to compensate for the low complexity library. SampleTag01 [GTTGTCAAGATGCTACCGTTCAGAGAAGAGTCGACTGCCATGTCCCCTCCGCGGGTCCGTGCCCCCCAAG] corresponds to WT cells and SampleTag02 [GTTGTCAAGATGCTACCGTTCAGAGACCGATTAGGTGCGAGGCGCTATAGTCGTACGTCGTTGCCGTGCC] to STAT6-KO cells.