OEX024230
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Transcriptomic single cell |
Transcriptomic single cell |
The renal biopsies were preserved in cold MACS Tissue Storage Solution (Miltenyi Biotec) and were minced into small pieces with a razor blade and incubated at 37°C in freshly prepared dissociation buffer containing enzymes from a Multi Tissue Dissociation Kit (Miltenyi Biotec) with a rotation speed of 200 rpm. Dissociated cells were harvested every 10 minutes by filtering the cell suspension through a 70‐µm cell strainer (FALCON) into 10% FBS buffer on ice. The residual biopsy tissue was dissociated again with 1 ml dissociation buffer for 10 minutes and passed through the cell strainer into the same FBS buffer from the first collection. This dissociation procedure was repeated three times until most of the tissue had been separated into single cells (total dissociation time was 30 minutes). Finally, the cells were collected by centrifugation at 1500 rpm for 5 minutes, resuspended in PBS containing 5% FBS, and strained through a 40‐µm cell strainer (FALCON) to further remove cell clumps and large fragments. Cell viability was approximately 90% for the biopsy used in this study, as assessed by Trypan Blue staining. Library construction was performed using the Chromium Single Cell 3′ Library & Gel Bead Kit v3.1 (10X Genomics) according to the manufacturer’s instructions. |
2023-06-12 |