RNA-seq of Homo sapiens: SK-N-BE(2) cells with TDP-43 knockdown - doxycycline 50 ng/ml - 3
SK-N-BE(2) 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 the following amounts of doxycycline hyclate (Sigma) and collected after 10 days: 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 75 ng/ml, 100 ng/ml, and 1000 ng/ml. 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).