title: NextSeq 500 sequencing; Time-course transcriptome analysis of a human neuronal cell line
library_construction_protocol: LUHMES cells were obtained from ATCC (https://www.lgcstandards-atcc.org/, CRL-2927). For routine maintenance, the vessels (typically T75 flasks) were pre-coated with first poly-L-ornithine hydrobromide (Sigma P3655, 50 µg/ml) and then human fibronectin (Sigma F1056, 1 µg/ml). The routine proliferation growth medium was DMEM/F-12 Ham (Sigma D6421) supplemented with L-glutamine (Sigma G7513, 2.5 mM), N-2 Supplement (Gibco 17502-048, 1X) and human basic Fibroblast Growth Factor (bFGF) (Thermo Fisher Scientific 68-8785-82, 20 ng/ml). For experiments, the vessels (typically 6-well plates) were pre-coated with higher concentrations of ornithine (100 µg/ml) and fibronectin (10µg/ml) to improve the cells attachment to the substrate. In experimental proliferation growth medium, 40 ng/ml bFGF was added instead of 20 ng/ml for a quicker propagation. The differentiation growth medium contains the experimental proliferation growth medium, but replaces the bFGF with tetracycline hydrochloride (Sigma T7660, 1 µg/ml). For the trypsinization step, we used a diluted trypsin-EDTA (0.25%, Thermo Fisher Scientific 25200056) with PBS in 1:1 volume. Total RNA was isolated from cells using QIAGEN RNeasy Mini Kit and DNase Set, following the RNeasy Mini Handbook instructions. The initial RNA concentration was measured with Nanodrop ND-100 (Thermo Fisher Scientific) and the quality was checked by an RNA integrity number > 8 (Agilent Tech 2200 Tape Station, Bioinformatics and Expression Analysis core facility at Karolinska Institutet). Prior to the transcriptomics library preparation, the RNA concentration was measured by Qubit 3.0 Fluorometer (Thermo Fisher Scientific) to dilute the RNA to 10ng/µl in 15µL nuclease free-water. 2 µl of lysate was used to make 48-plex RNA-seq libraries, which were prepared using modified STRT method with unique molecular identifiers (UMIs). (Islam et al. Genome Res. 2011 and Islam et al. Nat Methods. 2014). In order to remove amplification artefacts at high copy numbers, we have used longer UMIs of 8 bp.