GSM8788005: vascularized adipose organoid-2-3; Homo sapiens; RNA-Seq
RNA extraction from organoids was performed following the manufacturer's protocols (Isolation of RNA from organoids and spheroids, TFS-Assets/BID/Reference-Materials/isolation-rna-organoids-spheroids-white-paper.pdf). Briefly, samples processed with TRIzol Reagent (Invitrogen) were immediately flash-frozen in a mixture of dry ice and ethanol after cell lysis and stored at –80°C for RNA extraction at a later time. The purified total RNA was eluted in RNase-free water and stored at –20°C for subsequent analyses DTT, dNTP, RNase Inhibitor, RT Enzyme, and 5x RT Buffer were added to synthesize single-stranded cDNA. Exonuclease I and 10x Exonuclease I Buffer were used to remove single-stranded DNA, followed by magnetic separation. Poly(A) RNA was removed using Elution Buffer, and magnetic separation was performed again. Random primers, 10x Reaction Buffer, enhancer, dNTP, and cDNA Extend Polymerase I were used for two-strand cDNA synthesis. After removing the supernatant, Elution Buffer was added, and DNA beads were used to recover fragments. PCR amplification was performed with 2x HIFI Amplification Mix and Index, followed by examination of DNA beads for library fragments.