RNA-sequencing using SLAM-sequencing protocol of human iPSC-derived lower motor neurons treated with BDNF for 1, 2, 6 h
SLAM-sequencing was performed on i3 LMNs following protocols adapted from Herzog et al. (2017). Three biological replicates of i3 LMNs were treated with 100 µM 4sU (Control) + labelling medium or 100 µM 4sU + BDNF (BDNF condition) labelling medium on Day 7 for 1h, 2h, and 6h. 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. The samples were processed on RNeasy columns to re-isolate RNA. Sequencing libraries were prepared using the KAPA HyperPrep Kit with RiboErase kit (Roche). Samples were sequenced at 2 x 100 base pairs on an Illumina NextSeq 2000 machine.