GSM5494271: Day 8 Organoids; Homo sapiens; RNA-Seq
Organoid and chip samples (non-perfused and perfused) were collected and dissociated using TrypLE for 6 to 7 minutes at 37 degrees Celsius and with mechanical agitation to produce single cells. The TrypLE was neutralized using DMEM with 20% FBS. The single cells were spun down and washed twice with PBS before filtering and loading into the 10X Genomics Chromium Single Cell 3' Kit. Library preparations for the scRNA-seq was performed using 10X Genomics Chromium Single Cell 3' Kit, v3 (10X Genomics, Pleasanton, CA, USA). The cell count and the viability of the samples were accessed using LUNA dual fluorescence cell counter (Logos Biosystems) and a targeted cell recovery of 6000 cells was aimed for all the samples. Post cell count and QC, the samples were immediately loaded onto the Chromium Controller. Single cell RNAseq libraries were prepared using manufacturers recommendations (Single cell 3' reagent kits v3 user guide; CG00052 Rev B), and at the different check points, the library quality was accessed using Qubit  (ThermoFisher) and Bioanalyzer (Agilent). With a sequencing coverage targeted for 50,000 reads per cell, single cell libraries were sequenced either on Illumina's NovaSeq 6000 or NovaSeq 500 platforms using paired-end sequencing workflow and with recommended 10X; v3 read parameters (28-8-0-91 cycles).