RNA-seq of Homo sapiens: iPSC-derived cortical neurons with TDP-43/UPF1 knockdown - TDP-43 control UPF1 knockdown - 2
Human iPS cells used in this study were engineered (Fernandopulle et al., 2018; Wang et al., 2017) to express mouse or human neurogenin-2 under a doxycycline-inducible promoter, as well as an enzymatically dead Cas9 (+/? CAG-dCas9-BFP-KRAB) (Tian et al., 2019). To achieve knockdown, sgRNAs targeting either a non-targeting control guide (GTCCACCCTTATCTAGGCTA), UPF1 (GGCCAGACGCAGACGCCCCC), or TARDBP (GGGAAGTCAGCCGTGAGACC) were delivered to iPS cells by lentiviral transduction (Lois et al., 2002), for a total of 4 groups: Control for TDP-43 and UPF1 knockdown (control/control), Control for TDP-43 knockdown and positive for UPF1 knockdown (control/UPF1KD), Positive for TDP-43 knockdown and control for UPF1 knockdown (TDP43KD/control), Positive for TDP-43 and UPF1 knockdown (TDP43KD/UPF1KD). After lentiviral transduction, cells were plated in tissue culture dishes coated with human embryonic stem cell-qualified Matrigel (Corning). The following morning, cells were washed with PBS and the media was changed to E8 alone or (depending on cell number) E8 supplemented with 10 M ROCK inhibitor in a 37C, 5% CO2 incubator. Cells were passaged with accutase (Life Technologies) for 510 min at 37 C and then washed with PBS before re-plating. Two days after lentiviral delivery, cells were selected overnight with either puromycin (10 g/ml) or blasticidin (50100 g/ml). iPS cells were then expanded for 2 days before initiating neuronal differentiation. To initiate neuronal differentiation, 2025 million iPS cells per 15 cm plate were individualised using accutase on day 0 and re-plated onto Matrigel-coated tissue culture dishes in N2 differentiation media containing: knockout DMEM/F12 media (Life Technologies Corporation) with N2 supplement (Life Technologies Corporation), 1 GlutaMAX (Thermofisher Scientific), 1 MEM nonessential amino acids (Thermofisher Scientific), 10 M ROCK inhibitor (Selleckchem) and 2 g/ml doxycycline (Clontech). Media was changed daily during this stage. On day 3 pre-neuron cells were replated onto dishes coated with freshly made poly-L-ornithine (0.1 mg/ml; Sigma), 6-well dishes (2 million per well), in i3Neuron Culture Media: Brain-Phys media (Stemcell Technologies) supplemented with 1 B27 Plus Supplement (ThermoFisher Scientific), 10 ng/ml BDNF (PeproTech), 10 ng/ml NT-3 (PeproTech), 1 g/ml mouse laminin (Sigma), and 2 g/ml doxycycline (Clontech). i3Neurons were then fed three times a week by half media changes. i3Neurons were then collected on day 7 after the addition of doxycycline. RNA  was  extracted using  the  RNeasy  mini  kit  (Qiagen)  following  the  manufacturers  protocol,  including  the on-column  DNA  digestion  step.  RNA  concentrations  were  measured  by  Nanodrop  and  1,000  ng of  RNA  was  used  for  reverse  transcription. Sequencing libraries were prepared with polyA enrichment using a TruSeq Stranded mRNA Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 machine at UCL Genomics (2x150bp,  > 40 million reads per sample).