3500 genetic analyser

The human glioblastoma multiforme (GBM) cell lines A172 (ECACC 880624218), U251MG (ECACC 89081403; formerly known as U373MG), T98G (ECACC 92090213) and LN229 (ATCC-CRL-2611) were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) or the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as described before [26 (link)]. Fast and slowly-migrating native human GBM cells were isolated as mentioned previously [26 (link)] and in accordance with the Helsinki Declaration of 1975 with approval of the ethics committee of the University of Kiel, Germany, after written informed consent of donors (file reference: D 408/14). For an overview of clinical data available for these samples, please refer to [26 (link)]. Different GBM cells were checked for purity by immunostaining with markers specific for GBM cultures (glial fibrillary acidic protein (GFAP) and fibronectin [37 (link),38 (link)]; compare Figure S2) and for the absence of Mycoplasma contamination. LOX melanoma cells were a gift from Udo Schumacher, Department of Anatomy, University of Hamburg, Germany [39 (link)]. Cell lines’ identity was proven routinely by short tandem repeat profiling at the Department of Forensic Medicine (Kiel, Germany) using the Powerplex HS Genotyping Kit (Promega, Madison, WC, USA) and the 3500 Genetic Analyser (Thermo Fisher Scientific, Waltham, MA, USA).

PCR was performed using purified genomic DNA, AmpliTaq Gold 360 DNA polymerase and appropriate primers (Table S5). PCR amplicons were purified using a FastGene Gel/PCR Extraction Kit (NIPPON Genetics). Sequencing reactions were performed using purified DNA fragments, a BigDye Terminatorv3.1 Cycle sequencing Kit (Thermo Fisher Scientific) and appropriate primers (Table S5). A 3500 Genetic Analyser (Thermo Fisher Scientific) was used for Sanger sequencing analysis.

The complete FLCN gene coding region including adjacent intronic sequences was screened for mutations by PCR and subsequent Sanger sequencing following standard protocols (for primer details and PCR protocols see S1 Table). For each PCR 50–100 ng DNA were amplified using the HotStarTaq DNA Polymerase and Invitrogen^TMTaq DNA Polymerase recombinant (Qiagen, Hilden, Germany; Thermo Fisher Scientific, Dreieich, Germany). Purification of the amplification products was performed with the Qiagen PCR purification kit (Qiagen, Hilden, Germany), and PCR products were sequenced using the 3500 Genetic Analyser (Thermo Fisher Scientific, Dreieich, Germany). MLPA (multiplex ligation-dependent probe amplification) was performed using the SALSA MLPA P256 FLCN probemix (MRC Holland, Amsterdam, The Netherlands) according to the manufacturer’s protocol on the ABI 3100 Avant (Applied Biosystems, Darmstadt, Germany) and analyzed by Coffalyser.Net software (MRC Holland, Amsterdam, The Netherlands). Statistical analyses were performed using the two-tailed Mann-Whitney U Test and the Chi-square Test.

GWAS results were evaluated by two independent genotyping assays, the high-resolution melting (HRM) analysis and TaqMan test. HRM analysis was performed using MeltDoctor master mix (ThermoFisher Scientific, Waltham, MA, USA) and appropriate oligonucleotide primers (Eurofins Scientific, Luxembourg, Luxembourg) on a 7500 Fast Real-Time PCR System (ThermoFisher). For segment sequencing, a 3500 Genetic Analyser (ThermoFisher) was used. Genotyping using TaqMan assays (ThermoFisher) was also conducted on a 7500 Fast Real-Time PCR System.

For evaluating if the splicing process is different among the three genotypes for
rs28360071, a pair of primers on exon 3 (Forward: 5’-GGAGCAGGACCAGCTGATGTAT-3’)
and exon 4 (Reverse: 5’-TTTCTGCAATGGTGTCCAAGC-3’) of XRCC4 cDNA
was designed. After that, cDNA samples from five random patients (two carrying
II, one carrying ID (Insertion/Deletion. Heterozygous genotype of rs28360071)
and two carrying DD (Deletion/Deletion. Homozygous genotype of deletion allele
of rs28360071) genotypes) were amplified by PCR. The amplified fragment
resulting from the splicing process was expected as a 175 bp fragment (Figure
S3b). PCR fragments were purified using Agencourt AMPure XP magnetic beads
(Beckman Coulter Company, MA, USA), and sequenced with the BigDye Terminator
v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc.) on a 3500 Genetic
Analyser (Thermo Fisher Scientific Inc.). Experiments were performed in
triplicates. Representative chromatograms are shown in Figure S3.