title: GSM7502695: 48 hr DOX 5 uM, hiPSC-cardiac spheroid, replicate 3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated from hiPSC-CSs treated with DMSO, UF DOX (5 µM), and SPEDOX-6 (5 µM UF DOX equivalent) using a RNeasy Mini Kit (QIAGEN) with on-column DNase digestion. Nine spheroids were treated per condition for 48 hours at 40,000 cells per spheroid, following previously published ratios of hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs (Arzt et al., 2023). Each spheroid was washed with 200 μL PBS and then followed by a wash with 350 μL Buffer RLT containing B-mercaptoethanol and DTT to lyse the cells. Still in Buffer RLT, 3 spheroids from 1 condition were then transferred to an Eppendorf tube to generate 3 replicates of 3 spheroids per condition, or 120,000 cells per replicate. The contents of each tube were pipetted up and down and centrifuged at 8000 x g for 3 minutes. After discarding each supernatant, another 350 μL 70% ethanol was added per tube and the contents were transferred to RNeasy MinElute spin columns inside 2 mL collection tubes which were centrifuged for 15 seconds at 8000 x g. Buffer RW1 was then added to each column at 350 μL and spun for 15 seconds at 8000 x g. A mixture of Buffer RDD and DNase 1 was then added to each column at 80 μL and incubated at room temperature for 15 minutes. An additional 350 μL Buffer RW1 was added to each collection tube and centrifuged at 8000 x g for 15 seconds. A final addition of 500 μL 80% ethanol was added to each column and centrifuged at 8000 x g for 2 minutes. After transferring the spin column to a new collection tube, each column was spun at 5 minutes at full speed. The columns were transferred to a 1.5 mL tube and 14 μL RNase free water was added to the center of each column followed by centrifugation at full speed for 1 minute to elute the RNA. After a verification of the RNA yield of each tube via a NanoDrop 2000 Spectrophotometer (ThermoFisher), the samples were submitted to the Cedars-Sinai Genomics Core. RNA integrity was checked with an Agilent 2100 Bioanalyzer and only samples with an RNA integrity (RIN) score >9 were selected for cDNA library construction. Libraries were prepared with SMART-Seq v4 Ultra Low Input RNA Kit (Takara) and sequenced on a NextSeq 500 (Illumina) using single-end 75 bp reads at the Cedars-Sinai Genomics Core.