title: GSM9229529: Lungs from Gprc5a-/- mouse 24 at 3 months post-saline; Mus musculus; RNA-Seq
library_construction_protocol: Murine lung single cells were  stained with a cocktail of antibodies (1:250 each) against CD45-PE/Cy7 (103114, BioLegend), Icam2-A647 (A15452, Life Technologies), Epcam-BV421 (118225, BioLegend), and Ecad-A488 (53-3249-80, eBioscience). Stained cells were then washed, filtered using 40 μm filters, stained with SYTOX Blue and processed on a FACS Aria I instrument. Doublets and dead cells were eliminated, and viable (SYTOX-negative) epithelial singlets were collected in PBS containing 2% FBS. Cells were washed again to eliminate ambient RNA, and a sample was taken for counting by Trypan Blue exclusion before loading on 10XGenomics Chromium microfluidic chips. Spatial transcriptomic (ST) profiling of FFPE tissues of three Gprc5a-/- mouse samples was performed using the Visium platform from 10X Genomics according to manufacturer's recommendations and as previously reported by our group. Up to 10,000 cells per sample were partitioned into nanoliter-scale Gel beads-in-emulsion (GEMs) using Chromium Next GEM Single Cell 5' Gel Bead Kit v1.1 (1000169, 10XGenomics, ) and by loading onto Chromium Next GEM Chips G (1000127, 10XGenomics, Pleasanton, CA). GEMs were then recovered to construct single-cell gene expression libraries using the Chromium Next GEM Single Cell 5' Library kit (1000166, 10XGenomics) according to the manufacturer's protocol. Briefly, recovered barcoded GEMs were broken and pooled, followed by magnetic bead clean-up (Dynabeads MyOne Silane, 37002D, Thermo Fisher Scientific). 10X-barcoded full-length cDNA was then amplified by PCR and analyzed using Bioanalyzer High Sensitivity DNA kit (5067-4626, Agilent). Up to 50 ng of cDNA was carried over to construct gene expression libraries and was enzymatically fragmented and size-selected to optimize the cDNA amplicon size prior to 5' gene expression library construction. Further, samples were subject to end-repair, A-tailing, adaptor ligation, and sample index PCR using Single Index Kit T Set A (2000240, 10XGenomics) to generate Illumina-ready barcoded gene expression libraries. Library quality and yield was measured using Bioanalyzer High Sensitivity DNA (5067-4626, Agilent) and Qubit dsDNA High Sensitivity Assay (Q32854, Thermo Fisher Scientific) kits. Indexed libraries were normalized by adjusting for the ratio of the targeted cells per library as well as individual library concentration and then pooled to a final concentration of 10 nM. Library pools were then denatured and diluted as recommended for sequencing on the Illumina NovaSeq 6000 platform. For ST profiling, regions of interest per tissue/sample, each comprising a 6.5 x 6.5 mm capture area, were selected based on careful annotation of Hematoxylin and Eosin (H&E)-stained slides that were digitally acquired using the Aperio ScanScope Turbo slide scanner (Leica Microsystems Inc., Buffalo Grove, IL). HALO software (Indica Labs, Albuquerque, NM) was used for pathological annotation (tumour areas, blood vessels, bronchioles, lymphoid cell aggregates, macrophages, muscle tissue, normal parenchyma, and reactive pneumocytes) of H&E histology images. Spot-level histopathological annotation visualization was generated using loupe browser (v6.3.0). Briefly, cloupe file generated from Spaceranger was loaded into loupe browser along with the annotation table file , the annotation visualization was then generated in svg format using the export plot tool. Spatial transcriptomic RNA-seq libraries were generated according to manufacturer's instructions, each with up to ~3,600 uniquely barcoded spots. Libraries were sequenced on Illumina NovaSeq 6000 platform to achieve a depth of at least 50,000 mean read pairs/spot and at least 2,000 median genes/spot For scRNA-seq analysis of lung tissues from Gprc5a-/- mice, lung single cells were stained with a cocktail of antibodies (1:250 each) against CD45-PE/Cy7 (103114, BioLegend), Icam2-A647 (A15452, Life Technologies), Epcam-BV421 (118225, BioLegend), and Ecad-A488 (53-3249-80, eBioscience) to sort out epithelial and non-epithelial cells. Stained cells were then washed, filtered using 40 μm filters, stained with SYTOX Blue and processed on a FACS Aria I instrument. Doublets and dead cells were eliminated, and viable (SYTOX-negative) epithelial singlets were collected in PBS containing 2% FBS. Cells were washed again to eliminate ambient RNA, and a sample was taken for counting by Trypan Blue exclusion before loading on 10X Genomics Chromium microfluidic chips. Cells from Gprc5a-/-; SftpcCreER/+; RosaSun1GFP/+ mice were processed similarly except for sorting by GFP. Spatial transcriptomic (ST) profiling of FFPE tissues was performed using the Visium platform from 10X Genomics according to manufacturer's recommendations.