title: RNA-sequencing using SLAM-sequencing protocol of human iPSC-derived lower motor neurons treated with BDNF for 1, 2, 6 h
design: RNA-sequencing using SLAM-sequencing protocol of human iPSC-derived lower motor neurons treated with BDNF for 1, 2, 6 h
library_construction_protocol: The human induced pluripotent stem cells used are the WTC11 line, stably expressing a doxycycline-inducible hNIL construct and CRISPR/cas9. Briefly, iPSCs were seeded on tissue culture dishes coated with 120-180 μg/ml Geltrex (Thermo Fisher). Cells were maintained in iPSC medium containing Essential 8 Flex Medium (E8F, Thermo Fisher), E8 Flex supplement (Thermo Fisher), and 100 U/ml Pen/Strep and maintained in a 37 ºC, 5% CO2 incubator. Medium change was performed every 1-2 days. Passage of cells were performed with Stem Pro Accutase (Thermo Fisher) for 1-2 min at 37 ºC. Accutase was removed by centrifugation at 300 g for 5 min, and the cells were reseeded in iPS cell media supplemented with 10 μM ROCK inhibitor (Y-27632, Selleckchem) to facilitate survival. ROCK inhibitor was removed after 24 h. Cells were washed with PBS and RNA was extracted using the Qiagen RNeasy isolation and purification kit. RNA concentration was measured, and after equilibrating the amount of RNA in each sample, iodoacetamide (10 mM), NaPOH4 (pH 8, 50 mM), and DMSO (50% v/v) were added to each sample and incubated at 50 C for 15 min to facilitate the thiol-alkylation of 4sU.  Sequencing libraries were prepared using the KAPA HyperPrep Kit with RiboErase kit (Roche).