title: Time-course transcriptome analysis of a human neuronal cell line with RFX2 KO
design: Time-course transcriptome analysis of a human neuronal cell line with RFX2 KO
library_construction_protocol: A frozen vial of Lund human mesencephalic (LUHMES) cells, an immortalized dopaminergic neuronal precursor cell line, was purchased from the ATCC (https://www.atcc.org/products/crl-2927). LUHMES cells were cultured in a standard incubator (37°C, 5% CO2), following the same conditions previously described (Scholz et al., 2011; Lauter et al., 2020). LUHMES cells were grown in DMEM/F-12 Ham growth medium (Sigma-Aldrich D6421) supplemented with L-glutamine solution (Sigma-Aldrich G7513; 2.5 mM), N-2 supplement (Gibco 17502-048; 1×) and human heat stable basic Fibroblast Growth Factor (bFGF) (Thermo Fisher Scientific PHG0369; 20 ng/ml). For routinary maintenance, 2×106 cells were seeded in T75 flasks (Sarstedt 83.3911.002) pre-coated with poly-L-ornithine hydrobromide (Sigma-Aldrich P3655, 50 μg/ml) and fibronectin from human plasma (Sigma-Aldrich F1056; 1 μg/ml), in order to reach 80% confluency in three days and being then passaged using trypsin-EDTA (Gibco 25300054) diluted 1:1 in phosphate-buffered saline (PBS) (Sigma-Aldrich P4417) as cell dissociation reagent. For all the experiments, a higher concentration of ornithine (100 μg/ml), fibronectin (10 μg/ml) and bFGF (40 ng/ml) was used to improve cell adhesion and propagation. To differentiate LUHMES into postmitotic neurons, the bFGF in the growth medium was replaced with tetracycline hydrochloride (Sigma-Aldrich T7660; 1 μg/ml) in order to terminate the v-myc transgene expression. Total RNA was extracted from LUHMES cells and purified in a column using an RNeasy mini kit (Qiagen 74104) following the manufacturer's protocol. RNA integrity was checked on 1% agarose gel electrophoresis (Sigma-Aldrich A9539) and concentration and purity were assessed with a Nanodrop ND-100 (Thermo Fisher Scientific). For library preparation and transcriptome analysis, an additional DNase step (Qiagen 79254) was introduced during the total RNA isolation, the RNA integrity value was evaluated using an Agilent Tech 2200 Tape Station (RIN value >8. KI Bioinformatics and Expression Analysis core facility; https://ki.se/en/bionut/bea-core-facility) and the RNA concentration was measured with a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). The samples were then diluted to 10 ng/μl (±0.5 ng/µl) in nuclease-free water before proceeding with the RNA-seq library preparation.  We used 20 ng of RNA to generate two 48-plex RNA-seq libraries employing a modified STRT method with unique molecular identifiers (UMIs) (Islam et al. Genome Res. 2011, Islam et al. Nat Methods. 2014, Ezer et al. STAR Protoc. 2021). Briefly, RNA samples were placed in a 48-well plate, and a universal primer, template-switching oligonucleotides and a well-specific 6-bp barcode sequence (for sample identification) were added to each well of the plate (Krjutškov et al., 2016). The synthesized cDNAs from these samples were then pooled into one library and amplified by single-primer PCR using a universal primer sequence.