title: GSM4555852: Mtb_bcg_zdc_d15_1; Mus musculus; RNA-Seq
library_construction_protocol: Lung single-cell suspensions from vaccinated or Mtb-infected mice were isolated as previously described (Griffiths et al., 2016). Briefly, mice were euthanized with CO2 and lungs were perfused with heparin in saline. Lungs were minced and incubated in Collagenase/DNAse for 30 minutes at 37°C. Lung tissue was pushed through a 70 µm nylon screen to obtain a single cell suspension. Red blood cells were lysed and the cells were resuspended in suitable media or buffer for further use. Isolated total lung single cell suspensions were enriched for live cells using dead cell depletion kit according to manufacturer's instruction (Milteny Biotec) and subjected to droplet-based massively parallel single-cell RNA sequencing using Chromium Single Cell 5' (v3) Reagent Kit as per manufacturer's instructions (10x Genomics). Briefly, cell suspensions were loaded at 1,000 cells/uL with the aim to capture 10,000 cells/lane. The 10x Chromium Controller generated GEM droplets, where each cell was labeled with a specific barcode, and each transcript labeled with a unique molecular identifier (UMI) during reverse transcription. The barcoded cDNA was isolated and removed from the BSL-3 space for library generation. The cDNA underwent 11 cycles of amplification, followed by fragmentation, end repair, A-tailing, adapter ligation, and sample index PCR as per the manufacturer's instructions. Libraries were sequenced on a NovaSeq S4 (200 cycle) flow cell, targeting 50,000 read pairs/cell.
attributes.tag: GEO Accession
attributes.value: GSM4555852