GSM5702732: EC_cortex_WT_pig blood; Sus scrofa; RNA-Seq
After perfusion, tissue from cortex and medulla region of kidney were surgically dissected. The dissected tissue was weighted, placed on a petri dish containing ice-cold digestion HBSS, and cut into small pieces with a scalpel. Samples were then transferred into a 50 ML falcon tube containing 10 ML digestion buffer (HBSS (cat) supplemented with 0.2% Collagenase Type I (Sigma-Aldrich, Cat#C9891) and 15 mg/mL DNase I  and incubated at 37 Celsius degrees in a water bath for 40 min, shaking every 10 min. At the end of incubation, 20 ML ice cold HBSS buffer was added to the digested tissue suspension.     Cell suspension from cortex was filtered with 100 µm cell strainer and then filtered directly in the 40 µm cell strainer. Glomeruli were collected from the 40µm cell strainer with MACS buffer (PBS with 0.5% BSA, 2mM EDTA),  centrifuged at 300g for 5 min to collect the pellet, and then digested in 3 ML digestion buffer in 37 Celsius degree water bath for another 20 min, shaking every 10 min. The cell suspension of the flow-through and digested glomeruli were washed twice individually with MACS buffer, pooled together, resuspended in 400ul MACS buffer, and proceed to the MACS purification and FACS sorting with CD31.    Cell suspension from medulla was filtered with 70 µm cell strain first and then immediately 40 µm cell strain. The flow-through was centrifuged at 300g for 5 min to collect the pellet. Cell pellet was washed twice by MACS buffere and resuspended in 400ul MACS buffer, and proceed to the MACS purification. The 3' expression library was generated with Chromium Single Cell 3' Library, Gel Dead & multiplex kit and Chip Kit (v3, 10x Genomics). scRNAseq