title: GSM8249217: b342cor_min40_na; Homo sapiens; RNA-Seq
library_construction_protocol: For each sample tested, approximately 20 organoids were selected for staged partial dissociation along with 10 organoids which were fully dissociated in one step. Both populations were placed in 1.5mL Eppendorf tubes and suspended in 0.5mL of 30U/mL papain (Worthington) and 120U/mL DNase (Worthington) in Earle's balanced salt solution (EBSS) (Worthington). These tubes were then placed in a rotating incubator at 37°C. Every 20 minutes, the organoids being partially dissociated were allowed to settle, after which the supernatant containing liberated cells were collected. These organoids were washed once with papain and then placed in fresh papain solution for continued dissociation. The cells in the supernatant were passed through a 100 µm filter and counted using a Moxi Go II coulter counter (Orflo). After 60 minutes of total dissociation, any remaining solid aggregates in both the experimental and control tubes were fully dissociated via trituration (i.e., mechanical aspiration and expulsion via a 1mL pipette), counted, pelleted and prepared for scRNA-seq. All samples were pelleted and resuspended in 8µg/mL recombinant albumin (New England Biolabs) in dPBS[-/-] for encapsulation with the Chromium X instrument (10X Genomics). Approximately 8,000 cells were targeted for encapsulation per sample. Single cells were partitioned and barcoded with the Chromium Controller instrument (10X Genomics) and Single Cell 3' Reagent (v3.1 chemistry) kit (10X Genomics) according to the manufacturer's specifications with no modification (Rev E). Final libraries were quantified using the Qubit dsDNA HS Assay Kit (Life Technologies) and diluted to 3ng/µL in buffer EB (Qiagen). Library quality was confirmed using the Bioanalyzer High Sensitivity DNA Assay (Agilent) prior to sequencing.