GSM7428086: 262-B; Homo sapiens; RNA-Seq
Organoids were harvested, and a portion of the graft was flash frozen in liquid nitrogen. Human duodenal surgical sample was dissected and flash frozen in liquid nitrogen. Single nucleus extractions were performed on flash frozen organoids using the Minute Detergent Free Single Nucleus Isolation Kit (Invent Biotechnologies, Inc.). The manufacturer's instructions were followed, with a few modifications. Buffer A was supplemented with 100 Units of Protector RNAse Inhibitor (Roche), 1 μM DTT (Invitrogen), and 8x10-5 M Leptomycin B (Cell Signaling). Buffer B was supplemented with 110 Units of Protector RNAse Inhibitor and 8x10-5 M Leptomycin B. After the final spin in Buffer A, the nuclei were fixed in Riley's Buffer (PBS supplemented with 1% BSA,146 mM NaCl, 10 mM Tris-HCl, 1 mM CaCl2, 21 mM Mg Cl2, 20 Units of SUPERaseIn (Invitrogen), and 8x10-5 M Leptomycin B) supplemented with 0.1% PFA. After fixation, the nuclei were centrifuged at 500g for four minutes, resuspended in Riley's Buffer without PFA, and centrifuged again at 500g for four minutes. After centrifuging the nuclei in Buffer B as directed by the manufacturer's protocol, the nuclei were resuspended in Nuclear Storage Buffer (PBS supplemented with 1% BSA, 200 Units of Protector RNAse Inhibitor, and 8x10-5 M Leptomycin B). An aliquot of the nuclei was stained with Trypan Blue, and nuclear concentration was determined using a TC20 Automated Cell Counter. Nuclei were assessed for intactness using a Nikon Eclipse Ti prior to submission to the Single Cell Genomics Core at CCHMC for 10x sequencing. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics).