GSM4669483: Control_Sample_4; Homo sapiens; RNA-Seq
Human TS cells were transduced with shControl or shASCL2 lentiviral supernatant and incubated for 24 h. Media was changed 24 h post-transduction and selected with puromycin dihydrochloride (5mg/mL, cat. no. A11138-03, Thermo-Fisher) for two days. Cells recovered for 1-3 days in complete human TS medium prior to splitting for EVT differentiation. RNA was isolated on the eighth day of differentiation. Total RNA was isolated from cells and tissues using TRIzol reagent (cat no. 15596018, Thermo Fisher, Waltham, MA). Complementary DNA libraries from total RNA samples were prepared with Illumina TruSeq RNA preparation kits (Illumina, San Diego, CA). Barcoded cDNA libraries were multiplexed and sequenced with a HiSeq2000 DNA sequencer (100-bp paired-end reads) using a TruSeq 200-cycle SBS kit (Illumina) at the KUMC Genome Sequencing Facility. Reads from *.fastq files were mapped to the rat reference genome (Ensembl Rnor_5.0.78) using CLC Genomics Workbench 12.0 (Qiagen, Redwood City, CA). mRNA abundance was expressed in reads per kb of exon per million reads mapped.  A false discovery rate of 0.05 was used as a cutoff for significant differential expression (euglycemia vs hyperglycemia).