title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - knockdown 1hr 4sU - 2
design: 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. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100 M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2  250 bp) on an Illumina NovaSeq SP.