title: GSM8634140: ECM1 WT and KO; Mus musculus; OTHER
library_construction_protocol: Col1α2-Ecm1+/+ and Col1α2-Ecm1-/- mice ischemic kidneys were quickly collected and embed in OCT. Then, immersing the OCT-embedded block in isopentane precooled with liquid nitrogen.  Four 25um sections from each tissue block were used for total RNA extraction (Qiagen), and the percentage of RNA fragments larger than 200bp as determined by Agilent TapeStation 4200 High Sensitivity DNA ScreenTape (DV200 score) was used as a measure of RNA quality.  Tissue blocks with DV200 scores above 30% were used for downstream processing; selected tissues had DV200 scores above 97%. Briefly, 10um sections from each tissue block were placed on microscopy slides (ColorFrost Plus, Fisher) fixed with MeOH, H&E stained, then imaged in brightfield using a NanoZoomer SQ (Hamamatsu) slide scanner at 40x equivalent magnification (Protocol CG000614, 10x Genomics).  Each slide was incubated with mouse-specific probe sets provided by the manufacturer for subsequent mRNA labeling, probe transfer using the CytAssist (10x Genomics) onto one capture area of a 6.5mm x 6.5mm Visium CytAssist Slide, and subsequent library generation per the manufacturer's protocol (10x Genomics, CG000495, Rev C).  Library concentration was quantified using a Tapestation High Sensitivity DNA ScreenTape (Agilent) and fluorometry (Thermofisher Qubit) and verified via KAPA qPCR.  Libraries were pooled for sequencing on an Illumina NovaSeq X Plus 100 cycle 10B flow cell using a 28-10-10-90 read configuration, targeting 100,000 read pairs per spot covered by tissue (approximately 300M per tissue).