RNA-seq of Homo sapiens: SH-SY5Y cells with TDP-43 knockdown and cyclohexmide treatment - TDP-43 control no cycloheximide - 1
SH-SY5Y cells were transduced with a SmartVector lentivirus (V3IHSHEG_6494503) containing a doxycycline-inducible shRNA cassette for TDP-43. Transduced cells were selected with puromycin (1 ?g/ml) for one week, before being plated as single cells and expanded to obtain a clonal population. Cells were grown in DMEM/F12 + Glutamax (Thermo) supplemented with 10% FBS (Thermo) and 1% PenStrep (Thermo). For induction of shRNA against TDP-43, cells were treated with 25 ng/ml doxycyline hyclate (Sigma). After 10 days, cells were treated  either with 100 M cycloheximide (CHX) or DMSO (Pereverzev et al., 2015) for 6 h before isolating the RNA. Overall, 4 conditions were generated: Control for TDP-43 knockdown and DMSO (control/control), Control for TDP-43 knockdown and CHX-treated (control/CHX), Positive for TDP-43 knockdown and DMSO (TDP43/control) and Positive for TDP-43 knockdown and CHX-treated (TDP43/CHX). 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 (2x100bp, > 40 million reads per sample).