title: GSM5578194: O1_LacZ_4M_S5; Homo sapiens; RNA-Seq
library_construction_protocol: Organoids (LacZ, g2 and g3) were collected for single nuclei sequencing at 2 and 4 months. At harvest, organoids were dissected out of the Matrigel, frozen on dry ice and stored at -80°C. In order to reduce bias in the downstream analysis due to organoid heterogeneity, they were frozen 5 by 5 per time point and batch. The nuclei isolation protocol has been described in detail elsewhere (Sodersten et al., 2018). In brief, the organoids were thawed and homogenized in cold lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM DTT) using a tissue douncer (Wheaton). The homogenate was carefully layered on top of a sucrose solution (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl, pH 8.0, and 1 mM DTT) before centrifugation at 30,000 × g for 2 hours and 15 min. Pelleted nuclei were softened for 10 min in 100 ml of nuclear storage buffer (15% sucrose, 10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) before resuspended in 400 ml of dilution buffer (10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) and run through a cell strainer (70 mm). Cells were run through the FACS (FACS Aria, BD Biosciences) at 4°C with low flowrate using a 100 mm nozzle (reanalysis showed >99% purity), sorting 8,500 nuclei per sample which were then loaded onto 10X Genomics Single Cell 3' Chip along with the reverse transcription mastermix following the manufacturer's protocol for the Chromium Single Cell 3′ Library (10X Genomics, PN-120233) to generate single-cell gel beads in emulsion. cDNA amplification was done as per the guidelines from 10x Genomics and sequencing libraries were generated with unique sample indices (SI) for each sample. Libraries for samples were multiplexed and sequenced on a Novaseq using a 150-cycle kit using the recommended read length. Chromium Single Cell 3′ (10x) single nuclei RNA-seq