title: GSM6643279: Lung section derived from Sftpc:KrasG12D sample 2; Mus musculus; OTHER
library_construction_protocol: Spatial transcriptomic profiling was performed at the Molecular Genomics Core, a part of the Norris Comprehensive Cancer Center and under manufacturer's instructions. Briefly, two biological replicates (one block for each) of Gramd2:KrasG12D and Sftpc:KrasG12D underwent initial 10μM sectioning at the Tissue Pathology Core, part of the Norris Comprehensive Cancer Center. Sections were H&E stained (as above) and underwent pathology review. Regions of Interest (ROIs) then underwent sample preparation including test slide sample sequencing using the Visium Tissue Section Test Slide (PN-2000460, 10X Genomics, Dublin, CA, USA). RNA quality assessment of test slides was determined by first extracting RNA using the Qiagen RNeasy FFPE kit (#73504, Qiagen, Hilden, Germany) and measuring RNA concentration using a Qubit fluorometer (#Q33238, ThermoFisher Scientific, Waltham, MA, USA). To determine The percentage of total fragments >200nt in length was determined by running the samples using a 4200 Tapestation (#G2991AA, Agilent, Santa Clara, CA). Once samples were determined to be of high enough quality to continue, 5μM FFPE sections were then placed in a 42C water bath and once rehydrated adhered to the Visium Spatial Gene Expression Slide (PN-2000233, 10X Genomics). Subsequently, samples were dried at 42C for 3hrs in a desiccation chamber and underwent deparrafinization using Qiagen Deparaffinization Solution (Cat # 19093) at 60C for 2 hr. Subsequently H&E staining (#MHS16, #HT110116 Millipore Sigma, Burlington MA, USA) was performed according to Visium technical parameters (CG000409) and images were captured with a Zeiss Axioscan2 microscope using a 10x objective. Decrosslinking was performed according to 10X standard protocol (CG000407) and immediately hybridized to the Visium Mouse Transcriptome probe set V1.0, which contained 20,551 genes targeted by 20,873 probes. Post-probe extension, sequencing library construction was performed using unique sample indices using the Dual Index Kit TS, Set A (PN-1000251) for Illumina-compatible sequencing.