GSM9145002: Female Oxygen 1; Homo sapiens; RNA-Seq
Following 48-hour hyperoxia or room air exposure, organoids embedded in Matrigel were collected and immediately lysed in DNA/RNA shield (Zymo Research). Total RNA was extracted using the Quick RNA miniprep kit (#R1054, Zymo Research) according to manufacturer's instructions. RNA integrity was assessed using the Bioanalyzer 2100 system (Agilent Technologies, CA, USA) prior to library preparation. RNA library preparation was performed by Novogene. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, first strand cDNA was synthesized using random hexamer primers, followed by second strand cDNA synthesis using dUTP instead of dTTP to maintain strand specificity. The directional library was completed through end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Library quality was assessed using Qubit for quantification, real-time PCR for accurate quantification, and Bioanalyzer for size distribution detection.