RNA-seq analysis of human forebrain organoids irradiated with moderate to high doses of ionizing radiation
RNA-seq analysis of human forebrain organoids irradiated with moderate to high doses of ionizing radiation
Human forebrain organoids were irradiated and samples were lysed at 6 h, 24 h and 14 d following IR exposure. Organoid lysis was performed in RLT Plus lysis buffer (Qiagen, #74136) and samples were stored at -80Â°C. Total RNA extraction was performed by using the RNeasy Mini Kit (Qiagen, #74136). RNA quality was determined using the 5400 Agilent fragment analyzer system (Agilent Technologies, United States). Most of the samples had RNA integrity numbers >9.0. cDNA library construction and sequencing were performed by NovoGene (Cambridge, United Kingdom). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.