Bulk RNA-seq of primary mouse neural progenitor cells derived from the embryonic neocortex or medial ganglionic eminence at different time points after exposure to ionizing radiation
Bulk RNA-seq of primary mouse neural progenitor cells derived from the embryonic neocortex or medial ganglionic eminence at different time points after exposure to ionizing radiation
Mouse primary neural progenitor cells (NPCs) were irradiated and lysed at 6 h or 24 h following IR exposure. Cell lysis was performed in RLT Plus lysis buffer (Qiagen, #74136) and samples were stored at -80°C until further use. The prefrontal cortex (NCX) and medial ganglionic eminence (MGE) of the right hemisphere of C57Bl/6J mice at E13 were dissected and mechanically dissociated by gentle pipetting in Accutase (Stemcell Technologies, 07920). Primary mouse NPCs were cultured as monolayers onto Poly-D-Lysine (PDL) coated plates (Corning) in Dulbecco's Modified Eagle Medium (DMEM)/F-12 (Gibco, 11330-032) supplemented with B-27 (1:50, Gibco, 17504-044), N-2 (1:100, Gibco, 17502-048), Recombinant Human Fibroblast Growth Factor-basic (FGF-2, 20 ng/ml, Peprotech, 100-18C) and with or without Recombinant Murine Epidermal Growth Factor (EGF, 10 ng/ml, Peprotech, 315-09), respectively. Cells were passaged every 3 to 4 days (~80% confluent) by centrifugation at 300 x g for 3 min, followed by mechanical redissociation and replating as described above.  Acute irradiation (1 Gy) was performed using an X-strahl 320 kV (0.13 Gy/min, inherent filtration: 0.21 mmAl, additional filtration: 3.8 mmAl + 1.4 mm Cu + DAP, tube voltage: 250 kV, tube current: 12 mA, source distance: 100 cm, vertical beam orientation) in accordance to ISO 4037. For sham (0 Gy) irradiation, cells were moved to the irradiation facility but not placed under the X-ray beam. 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.