GSM9695020: Myeloid cells from Mtb-infected mice; Mus musculus; RNA-Seq
3 C57BL/6 mice were infected with 105 CFU of Lp-ΔflaA expressing an mCherry plasmid. After 3 days, these mice, along with 3 other naïve C57BL/6 mice, were subsequently infected with ~50 CFU of Mtb-mCherry. At 14 days post-infection, the lungs were harvested and processed as for flow cytometry, as described above. Samples were then stained on ice for 30 minutes with APC-labeled CD64 (X54-5/7.1, BioLegend). Myeloid cells were magnetically enriched using an EasySep APC Positive Selection Kit II (StemCell Technologies) and MojoSort Magnets (BioLegend). Samples were then stained on ice for 45 minutes with the following anti-mouse antibodies in the presence of True-Stain Monocyte Blocker (BioLegend): TruStain FcX PLUS (S17011E, BioLegend), BV786-labeled CD45.2 (104, BioLegend), and Pacific Blue-labeled B220 (RA3-6B2, BioLegend), CD90.2 (53-2.1, BioLegend), and Ly6G (1A8, BioLegend). The Pacific Blue-labeled antibodies were used to exclude neutrophils, T cells, and B cells. Additionally, the following TotalSeq-A-labeled anti-mouse antibodies were added to detect protein expression in the scRNA-seq dataset: Ly6C (HK1.4, BioLegend), CD274 (MIH6, BioLegend), Siglec F (S17007L, BioLegend), CSF1R (AFS98, BioLegend), CD11b (M1/70, BioLegend), CD86 (GL-1, BioLegend), and MHC II (M5/114.15.2, BioLegend). A unique anti-mouse TotalSeq-A Hashtag antibody (1-6; BioLegend) was added to each sample to allow up to 6 samples to be mixed per lane on the 10X Chromium Next GEM Chip (10X Genomics). After staining, cells were resuspended in PBS with 5% Newborn Calf Serum and Sytox Blue Dead Cell Stain (Thermo Fisher Scientific). Cells were sorted using a 120 μm microfluidic sorting chip in a 4 laser SH-800 cell sorter (Sony) on the purity setting. Cells were collected in PBS containing 50% fetal calf serum. The v3.1 chemistry Chromium Single Cell 3' Reagent Kit (10X Genomics) was used to generate scRNA-seq libraries. The manufacturer protocol was followed, with the following minor modifications. Lanes of the 10X Chromium Next GEM Chip were super-loaded with 29,000 cells. During the loading step, 0.5 U/μL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) was added to single cell RT master mix. During the cDNA amplification, 1 μL of ADT and HTO additive primers (0.2 μM stock) were added as recommended by the CITE-seq and Cell Hashing Protocol[42]. After the purification step, samples were decontaminated by 2 rounds of centrifugation through a 0.2 μM filter microcentrifuge tubes and removed from the BSL3. cDNA libraries were prepared following the 10X Genomics protocol. The ADT and HTO libraries were prepared following the CITE-Seq and Cell Hashing Protocols. DNA quality was measured using a TapeStation (Agilent). The cDNA, ADT and HTO libraries were pooled at the following proportion: 80% cDNA, 10% ADT, and 10% HTO. The libraries were sequenced on an Illumina platform (Azenta).