GSM7505360: Mouse replicate 3 Nur77GFP HI; Mus musculus; RNA-Seq
Mycobacterium tuberculosis-infected Nur77-GFP mice were euthanized with CO2 and lungs were suspended in a digestion buffer containing DMEM, FBS, Collagenase D, DNase, Heparin, CaCl 2 , and MgCl 2 . Lungs were incubated at 37˚C for 30 minutes and dissociated with a gentleMACS dissociator (Miltenyi Biotec), according to the manufacturer's protocol. To isolate cells, lung suspensions were filtered through a 70 μm cell strainer. Sorting for CD44hi, CD4+, GFP HI and LO cells was performed with a Sony MA900 cell sorter. Cells FACS sorted (either GFP-HI or -LO) into DMEM with 40% FBS were used for scRNA-Seq sample preparation. After sorting, cell number was confirmed with the Luna II (Logos Biosystems). Approximately 100,000 cells from each population were sorted into DMEM-40 before resuspension in Tri reagent. Samples in TRI Reagent were centrifuged at 1000 x g for 10 minutes. Samples were resuspended in chloroform and incubated for 3 minutes then centrifuged at 12,000 x g for 15 minutes. The supernatant was collected, isopropyl alcohol added, incubated for 10 minutes, then centrifuged at 7,500 x g for 5 minutes. The supernatant was discarded then samples were air dried before resuspension in RNase-free water.