title: GSM9159731: Replica set S1, Library 1; Homo sapiens; RNA-Seq
library_construction_protocol: Organoids were recollected in a 15 mL polypropylene (Falcon) tube to wash with PBS 3 times. As a dissociation reagent, 1 mL of trypsin 0.5% — EDTA (10×) with 50 μM Y-27632, 40 U/μL RNase inhibitor, and 1 U DNase I was added to the organoids and kept on 37°C in a Vari-Mix Platform Rocker at 30 rpm for 15 minutes. Every 5 minutes, we mechanically disrupted the organoids until it was homogenized into single cells. Afterward, we proceeded to the inactivation of trypsin with 1 mL Advanced DMEM/F12 mix containing Bovine Albumin Fraction V (7.5% solution) 50 μM Y-27632, 40 U/μL RNase inhibitor, and 1U DNase I. We collected and filtered the cells trhough a cell strainer. We then add another 1 mL Advanced DMEM/F12 mix to recover any left cells from the strainer. We took 10 μL of single-cell medium mixed with 10 μL Trypan Blue (0.4%) to count the number of single cells to proceed with their fixation. We carried out the fixation of at least 1 × 105 required single cells, following the protocol described in the Evercode fixation kit (Parse Bioscience). The preparation for the cell barcoding and the posterior library for sequencing was performed with Evercode WT Mini, Chemistry v2 (Parse Bioscience). The Genomics Core facility of the University of Chicago performed the sequencing using a HiSeq Illumina platform, paired end.