Cd326 microbeads

To rapidly isolate RNA from 12.5 Gy and non-irradiated tissue for sequencing, we developed a technique of rapidly isolating pulmonary epithelia based on the expression of the epithelial cell-adhesion molecule (EpCam) CD326 (Fig. 1). To isolate CD326+ epithelium from whole lung-tissue digest, cells were pelleted and resuspended in microbead buffer (PBS, 0.5% BSA, and 2 mM EDTA) plus CD45 magnetic microbeads (Miltenyi Biotech #130 052 301, Auburn, CA) per manufacturer’s instructions to remove the CD45+ fraction of cells from the lung homogenate. This CD45-depleted population of cells was then recounted, pelleted, and resuspended in bead buffer plus CD326 microbeads (Miltenyi Biotech #130 105 958). Again, cells were washed in buffer solution, pelleted, and resuspended in 500 μl fresh buffer prior to a second magnetic separation using a Miltenyi LS column. Unbound CD326 negative cells were washed from the column, and CD326+ cells trapped within the column were flushed from the column and collected. Cells were pelleted, washed, and transferred into 600μl RLT Lysis buffer (Qaigen, Hilden, Germany) and stored at −80°C until processing for RNA isolation. All RNA isolation, quality-control analysis, and low-input RNA sequencing procedures were performed by the University of Rochester Genomics Research Center.

Kidney organoids were transferred to a drop of trypsin-EDTA, minced with a surgical blade, transferred to a 15-ml tube with 3 ml of trypsin-EDTA, and incubated at 37 °C for 10 min while the disintegrating organoid pieces were gently pipetted every 3 min. Next, 8 ml of DMEM + 10% fetal bovine serum was added and cells were pelleted by centrifugation (250g for 5 min). Then, the pellet was resuspended in 2 ml of epithelial cell culture medium (Renal Epithelial cell growth medium; Banksia Scientific). The remaining cell clumps were removed with a 40-µm cell strainer (BD Biosciences), and cells were counted. Cells (10⁷) were repelleted by centrifugation (250g for 5 min), resuspended in 200 µl of MACS buffer (46.7 ml PBS+; 3.3 ml of 7.5% BSA; 200 µl 0.5 M EDTA), mixed with 20 µl of CD326 microbeads (Miltenyi Biotec), and refrigerated to 4 °C. After 30 min, 5 ml of MACS buffer was added and cells were pelleted by centrifugation, resuspended in 500 µl MACS buffer, and applied to a prerinsed MS column in the MACS separator. After three washings with MACS buffer, cells were collected with 1 ml of MACS buffer after the column was removed from the MACS separator. This routinely yielded approximately 3 × 10⁵ EpCAM⁺ cells per organoid and live cell rates > 75%. The isolation of epithelial cellular fractions from patient organoids has been described previously^{30 (link)}.