Illumina NovaSeq 6000 paired end sequencing; Timecourse RNA-seq of mouse embryonic stem cells during neural differentiation
Timecourse RNA-seq of mouse embryonic stem cells during neural differentiation
To differentiate ESCs into neurons, we performed monolayer neural differentiation. ESCs were washed with PBS, dissociated using Accutase (StemPro ref. A1110501) and resuspended in N2B27 medium: 1:1 mix of Advanced DMEM/F-12 (Gibco ref. 12634010) and Neurobasal (Gibco ref. 21103049) supplemented with 1x L-Glutamine (Gibco ref. 25030024), 1x MEM non-essential amino acids (Gibco ref.11140035), 0.5x N-2 supplement (Gibco ref. 17502048), 0.5x B-27 Supplement (Gibco ref. 17504044) and 0.1mM 2-mercaptoethanol (Gibco ref. 31350010). The appropriate number of cells (100,000 cells per well of a 6-well plate) was transferred into gelatin-coated plates containing N2B27 medium. The medium was changed every 2 days until analysis. At the appropriate timepoint, cells were directly lysed on the plate and total RNA was extracted using the RNeasy Plus Mini kit (Qiagen), following manufacturerâ€™s instructions. Eventual contaminating genomic DNA was removed by DNase treatment. Equal amounts of RNA sequins (Garvan Institute of Medical Research, Australia) spike-in mix were added to each sample and RNA-seq libraries were prepared by polyA-enrichment using the NEBNext Ultra II Library Prep Kit (NEB ref. E7645) together with indexed adapters.