title: GSM5100067: Mtb_uninfected_cells_1; Mus musculus; RNA-Seq
library_construction_protocol: Three weeks post-infection mice were sacrificed, the lungs aseptically removed and placed in PBS containing 5% FBS. To minimize unwanted changes in the gene expression profile of both host and bacteria, samples were kept on ice and immediately processed using a GentleMACS tissue dissociator (Miltenyi Biotec)(Pisu et al., 2020b). The dissociated lung material was then passed through a 70mM cell strainer and red blood cells were lysed with ACK lysis buffer (Lonza). Bystander populations: Single cell suspensions from mice infected with smyc'::mCherry (n=3, 2 batches, total 6 mice) were incubated for 20 minutes in the dark with fluorophore-conjugated antibodies against CD45 (104, BD). Stained samples were washed twice in PBS, resuspended in sorting buffer, passed through a 40M strainer and sorted according to the gating strategy depicted in Figure S1B. Samples were maintained at 4C during sorting and collected directly into Cell Staining Buffer (Biolegend).  Uninfected populations: Single cell suspensions from uninfected healthy mice (n=3, 2 batches, total 6 mice) were incubated for 20 minutes in the dark with fluorophore-conjugated antibodies against CD45 (104, BD). Stained samples were washed twice in PBS, resuspended in sorting buffer, passed through a 40M strainer and sorted according to the gating strategy depicted in Figure S1C. Samples were maintained at 4C during sorting and collected directly into Cell Staining Buffer (Biolegend). Unstained uninfected mice were used to define the sorting gate for the CD45 signal. Sorted murine populations were stained with HTO and ADT antibodies (Biolegend) following published protocols (Stoeckius et al., 2017), with slight modifications. Briefly, sorted cells were spun down at 500g for 5', resuspended in 50mL of Cell Staining buffer containing 0.25mg of TruStain FcX PLUS (anti-mouse CD16/32 blocking reagent) (Biolegend) and incubated for 10' at 4oC. 50mL of an ADT + HTO antibody cocktail mix was then added to the samples and incubated for another 30' at 4C. After incubation, samples were washed 2X in cell staining buffer and differentially tagged samples (Hspx-high/Hspx-low and Bystander/Uninfected) mixed together and resuspended in DPBS 1X. Ice-cold methanol was then added drop-by-drop to a final concentration of 90% (V/V). Fixed samples were stored at -20oC O/N. After fixation, samples were taken out of the BSL3 facility, equilibrated on ice for 15', washed twice with Rehydration Buffer (1X DPBS containing 1.0 % BSA (Thermo-Fisher) and 0.5U/μl RNAse Inhibitor (Sigma)) and the amount of recovered cells quantified before loading into the 10X chip. For the mRNA library preparation, we followed the commercially available 10X protocol (CG000206 Rev D), with a slight modification in Step 2.2 and the addition of 1uL of ADT and HTO additive primers (0.2uM stock) as previously described. (Stoeckius et al., 2017) For both murine and human samples, HTO and ADT libraries were generated following Biolegend commercially available protocols.