title: scRNAseq of mus musculus lung neutrophils infected by mycobacterium bovis BCG
design: For the lung neutrophils cell sorting, 21 days after BCG infection, lung were digested and enriched in CD11b with a positive magnetic bead selection (Miltenyi, according to manufacturers instructions). CD11b+/Ly-6C+/Ly-6G+ neutrophils were sorted on a MoFlo AstriosEQ high speed cell sorter (Beckman Coulter) according to our previously published protocol (Rambault Doz-Deblauwe et al., Frontiers immunol). Neutrophils were recovered in complete medium and immediately processed for single cell RNAseq. Within 1 hour after sorting, cells were encapsulated with barcoded Single Cell 3 v3.1 Gel Beads and a Master Mix to form Gel Beads-in-emulsion using the 10X Genomics Chromium technology. Approximately 12 000 cells were used. The Single Cell 3 libraries were generated as recommended by the manufacturer (10x Genomics). The libraries were equimolarly pooled and sequenced (paired-end sequencing) using one lane of an Illumina NovaSeq6000 device (IntegraGen, France), yielding a total of 640 million reads. The data were intercepted to obtain Read1 and Read2 index files, I1 and I2, containing barcode and UMI sequence information.