GSM9125269: Lung CD45minus, HTO; Mus musculus; RNA-Seq
Lungs were collected from IM-DTR mice at 0, 12, 24, and 48 hours following interstitial macrophage depletion induced by intraperitoneal injection of 50 ng diphtheria toxin (DT), with three to four mice analyzed per time point. Single-cell suspensions were obtained after enzymatic digestion and non-hematopoietic CD45- niche cells were isolated by MACS using CD45 MicroBeads (MiltenyiBiotec, 130-052-301). Lung endothelial cells, epithelial cells and fibroblasts werethen FACS sorted as separately as CD45+CD31+EpCAM-, CD45-CD31-EpCAM+ and   CD45-CD31-EpCAM- cells, respectively. Sorted  lung endothelial cells, epithelial cells and fibroblasts were pooled at an equicellular ratio of 1:1:1. Cells were then barcoded per timepoint post-DT injection with TotalSeq™-A anti-mouse Hashtag antibodies (BioLegend). Hashtag barcoded cells were washed, pooled, spun down and resuspended in PBS with 0.04% UltraPure™ BSA (Thermo Fisher Scientific, AM2616) at a final concentration of 500 cells/μl. Cell suspensions were loaded into the Chromium Controller (10x Genomics) at a target recovery of 3 x 10³ cells per timepoint post-DT injection. Cells were encapsulated, partitioned, and their polyA RNAs captured and barcoded using Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). The cDNAs were amplified and libraries compatible with Illumina sequencers were generated using Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). For Hash Tag Oligonucleotide (HTO) library, 1 μl HTO additive primer v2 (0.2 μM stock) were added to the mix at the cDNA amplification step. The libraries were sequenced on an Illumina NovaSeq sequencer on an SP100 cell flow (read 1, 28 cy; read 2, 76 cy; index i7, 10 cy; index i5, 10 cy) with a sequencing depth of 20,000 reads per cell.