title: Bulk short- and long-read RNA-sequencing of iNeurons in Fragile-X and control cell-lines
design: Bulk short- and long-read RNA-sequencing of iNeurons in Fragile-X and control cell-lines
library_construction_protocol: NGN2-induced glutamatergic neurons were generated from Fragile X syndrome patient-derived induced pluripotent stem cells (iPSCs) and an isogenic CRISPR-corrected rescue line. Neuronal differentiation was performed for 21 days using doxycycline-inducible NGN2 expression. Cells were cultured on polyornithine/laminin coated plates and maintained with BDNF-containing neuronal medium. Cells were harvested at day 21 for downstream single-cell and bulk RNA sequencing. Total RNA was extracted from NGN2-induced neurons using TRIzol reagent according to the manufacturerâ€™s instructions. RNA concentration was measured using Qubit RNA High Sensitivity assays and RNA integrity was assessed using Agilent ScreenTape analysis. Samples used for sequencing had RNA integrity numbers (RIN) greater than 9. Library preparation was performed using 300 ng of total RNA (RIN > 7) as input. High-quality cDNA was generated using the Iso-Seq Express 2.0 Kit (PB, 103-071-500) and further processed with the Kinnex PCR 8-fold Kit (PB, 103-071-600) for multiplexing. Following ten cycles of cDNA amplification, libraries were purified using SMRTbell Cleanup Beads (PB, 102-158-300). Final library concentration and fragment size distribution were validated via Agilent TapeStation 4200 and Qubit Flex Fluorometer.