Bulk short- and long-read RNA-sequencing of iNeurons in Fragile-X and control cell-lines
Bulk short- and long-read RNA-sequencing of iNeurons in Fragile-X and control cell-lines
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. ng of total RNA, inclusive of spike-in controls, was prepared using the cDNA-PCR Sequencing V14 kit (SQK-PCS114.24). The protocol included 14 cycles of PCR amplification to generate sufficient material for nanopore sequencing.