GSM7851847: 2 Weeks Bystander, ADT; Mus musculus; OTHER
At 2, 4, and 6 weeks post-infection (w.p.i.), mice were euthanized. Lungs were aseptically removed and immersed in PBS containing 5% FBS and Collagenase IV (250U/mL). To preserve the gene expression profiles of both the host and bacteria, samples were immediately processed using a GentleMACS tissue dissociator (Miltenyi Biotec) and maintained on ice. The dissociated lung material was subsequently strained through a 70-µM mesh, and red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis buffer (Lonza). Cells from infected mice (n = 5/timepoint) were washed in PBS containing 5% FBS, resuspended in sorting buffer (PBS, 1% FBS, 5 mM EDTA, and 25 mM Hepes), filtered through a 40-µM strainer, and sorted. Throughout the sorting, samples were kept at 4°C and directly collected into Cell Staining Buffer (BioLegend). Sorted cells were centrifuged at 500 g for 5 min and then resuspended in 50 µl of cell staining buffer containing 0.25 µg of TruStain FcX PLUS (BioLegend), followed by a 10 min incubation at 4°C. An ADT plus HTO antibody cocktail mix (50 µl) was then added to the samples, and the cells were further incubated for 30 min at 4°C. After two washes in cell staining buffer, differentially tagged samples (e.g., hspx′::GFPhigh/hspx′::GFPlow) were combined and resuspended in 1× Dulbecco's PBS. The samples were then fixed by slowly adding ice-cold methanol to a final concentration of 90% (vol/vol) and stored at −20°C overnight. Post-fixation, the samples were brought out of the BSL3 facility, equilibrated on ice for 15 min, and washed twice with rehydration buffer (1× Dulbecco's PBS with 1.0% BSA [Thermo Fisher Scientific] and 0.5 U/µl RNase Inhibitor [Sigma-Aldrich]). The cell count was determined prior to loading onto the 10× chip. For mRNA library preparation, we adapted the 10× protocol (CG000206 Rev D), making a minor alteration in step 2.2. Specifically, we incorporated 1 μl of ADT and HTO additive primers (0.2 μM stock) following the method described by Stoeckius et al76. HTO and ADT libraries were prepared according to BioLegend's standard protocols. The mRNA, HTO, and ADT libraries underwent quality control assessment using an Agilent Fragment Analyzer. Their concentrations were determined using the QX200 digital PCR system from Bio-Rad. Libraries were pooled in the same sequencing run at specific ratios: 90% mRNA, 5% ADT, and 5% HTO. Sequencing was performed on the Nextseq 2k (50bp kit) or NextSeq500 (Illumina) using the 75-bp NextSeq kit. The cycle distribution was: read 1 (28 cycles), i7 index (8 cycles), and read 2 (55 cycles for nextseq 500 and 52 cycles for nextseq 2k). Sequencing depth exceeded 50,000 reads/cell.