GSM9258365: mln.uninf.d0.1; Mus musculus; RNA-Seq
Single cell suspensions were generated from medLN, blood, and lung samples as described above prior to Mtb infection and at days 0, 15 and 28 post-Mtb infection from pools of 8-10 mice for each suspension/condition. Cells were resuspended in 200 μl MACS buffer (PBS containing 2.5% FBS plus 1 mM EDTA), filtered through a 70 μm filter, and run on a FACS AriaII (BD) sorter. To collect parenchymal cells for single-cell RNA sequencing, alveolar macrophages (AM, SiglecF+ Autofluorescence+) were sorted separately into one collection tube to account for autofluorescence in the IV label channel, and all other IV-negative cells were sorted into another collection tube. After sorting AMs were included in respective lung samples (Mtb-infected or uninfected). Mtb-infected and uninfected bystander (mCherry+ and mCherry -) samples were harvested separately. After one round of washing with ice-cold DPBS, cells were resuspended to 1000 cells/μl in DPBS, and 8000 cells were inputted into the 10X Genomics pipeline following the manufacturer's recommendations. After the generation of cDNA following the manufacturer's protocol, samples were centrifuged through two sequential rounds of 0.2 μm SpinX (Costar) columns to sterilize the sample for removal from the BSL3 facility and subsequent library generation. Libraries were submitted to Psomagen (Rockville, MD) for NovaSeq sequencing, with 300M reads per sample