RNA-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown (30 days) - Control 2
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi  knockdown of TDP-43 in iPSCs was achieved  using  sgRNA  targeting  the  transcription  start  site  of  TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. i3Neurons  were  induced  as previously  described (Fernandopulle et al., Current Protocols in Cell Biology, 2018)  with  the  addition  of  SMAD  and  WNT  inhibitors  (Nehme et al., Cell Reports, 2018)(SB431542  10  ; LDN-193189  100  nM;  XAV939  2    all  from  Cambridge  Biosciences). After  induction,  cells were cultured in BrainPhys Media (StemCell  Technologies) with 20 ng/ml BDNF (PeproTech), 20 ng/ml GDNF (PeproTech), 1x N2 supplement (Thermo  Fisher), 1  x B27 supplement (Thermo Fisher), 200 nM Ascorbic Acid (Sigma), 1 mM dibutyryl cyclic-AMP (Sigma) and 1 /ml Laminin (Thermo  Fisher) as previously  described (Bardy et al., PNAS, 2015) and harvested 30 days after differentiation. Strand-specific, poly(A) enriched sequencing libraries dataset were prepared using the Kapa mRNA Hyper Prep kit. 100ng total RNA was used as input material for poly(A)+ mRNA capture. Fragmentation was performed for 6 minutes at 85 to obtain a target fragment size of 300-400bp, and 13 cycles of PCR amplification were performed. The resulting libraries were sequenced 2x150bp on the Illumina NextSeq2000 machine.