GSM3613433: Healthy donor 7; Homo sapiens; RNA-Seq
For each RNA preparation, 2 wells of organoids in expansion phase were collected in Trizol reagent (Sigma) 4-5 days after splitting. RNA was extracted according to manufacturer's instruction and resuspended in 30ul of nuclease free water. Total RNA was quantitated (ND1000 Spectrophotometer – PEQLAB). Samples were diluted accordingly to a mean concentration of approximately 100-150ng/μl and their quality assessed on a Bioanalyzer (Agilent Technologies) using the Agilent RNA 6000 Nano Kit reagents and protocol (Agilent Technologies). The quantity and quality of RNA samples from sorted cells were analyzed using the bioanalyzer form Agilent in combination with the Agilent RNA 6000 Nano. RNA samples with RNA Integrity Number (RIN) > 7 were further used for library preparation using the 3' mRNA-Seq Library Prep Kit Protocol for Ion Torrent (QuantSeq-LEXOGEN™) according to manufacturer's instructions. The quantity and quality of libraries were assessed using the DNA High Sensitivity Kit in the bioanalyzer, according to the manufacturer's instructions (Agilent Technologies). Libraries were subsequently pooled and templated using the Ion PI™ Hi-Q™ OT2 200 Kit (ThermoFisher Scientific) on Ion OneTouch 2 System. Sequencing was performed using the Ion PI™ Hi-Q™ Sequencing 200 Kit and Ion PI™ Chip Kit v3 (ThermoFisher Scientific) on an Ion Proton™ System, according to the manufacturer's instructions.