GSM7662593: PSC_6_CNT; Homo sapiens; RNA-Seq
ECO from PSC (n=4) and non-PSC patients (n=4), all at passage 3, were treated with vehicle or human recombinant IL-17A (100 ng/mL) for 24 hours. The organoids underwent trypsin digestion followed by mechanical dissociation to obtain single cell suspensions in PBS + 0.1% BSA and were submitted to the Gene Analysis Core of the Medical Genome Facility (Mayo Clinic, Rochester, MN) for scRNA-seq analysis. The cDNA master mix was prepared according to the manufacture's instruction for Chromium Next GEM Single Cell 3' Library and Gel Bead Kit (10x Genomics, Pleasanton, CA). The standard targeted cell recovery was set to ~3000 cells. The cell suspension and master mix thawed gel beads and partitioning oil were added to a Chromium Single Cell G chip. The filled chip was loaded into the Chromium Controller, where each sample was processed and the individual cells within the sample were partitioned into uniquely labeled GEMs (Gel Beads-In-Emulsion). The GEMs were collected from the chip and subjected to reverse transcription, GEM dissolution, and cDNA clean-up.  The resulting cDNA contained a pool of uniquely barcoded molecules. A portion of the pooled cDNA was used for library construction, where standard Illumina sequencing primers and a 10x Genomics unique i7 sample index were added to each cDNA pool. All cDNA pools and resulting libraries were measured using Qubit High Sensitivity assays (Thermo Fisher Scientific, Waltham, MA) and Agilent Bioanalyzer High Sensitivity chips (Agilent, Santa Clara, CA). Libraries were sequenced at between 40,000 and 50,000 fragment reads per cell following Illumina's standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit (Illumina, San Diego, CA). The flow cells were sequenced as 100 X 2 paired end reads on an Illumina HiSeq 4000 HD using HiSeq 3000/4000 sequencing kit and HCS v3.4.0.38 collection software. Base-calling was performed using Illumina's RTA version 2.7.7.