Bigdye v3.1 cycle sequencing kit

Genomic DNA and total RNA were extracted from excised tissue or cultured cells with the AllPrep DNA/RNA mini kit (Qiagen). Total RNA (1 μg) was used for the reverse transcription (RT) reaction with Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. The CIC–DUX4 fusion transcript was amplified with CIC forward primer CICF4120 (5′-TGAGTTGCCTGAGTTTCG-3′) and DUX4 reverse primer DUX4RTr2 (5′-TGAGGGGTGCTTCCAGCG-3′), using Q5 High-Fidelity DNA polymerase (New England Biolabs). For Sanger sequence analysis, the products were further amplified with forward (CIC2F; 5′-ATCATGCAGGCTGCCACT-3′) and reverse DUX4R2 (5′-ATGCCTTGCATCTGCCC-3′) primers for junction, or with reverse DUX4-R1 (5′-TCTAGGAGAGGTTGCGCCTG-3′) primer to determine whether the fusion gene was DUX4 (4q) or DUX4 L (10q), respectively. The PCR products were purified with ExoSAP-IT (Affymetrix) and direct sequencing was performed using BigDye v3.1 Cycle Sequencing Kit (Applied Biosystems) on the Applied Biosystems 3130xL by Eurofin genomics (Japan). The sequence data were matched against the CIC (NM_015125.4) and DUX4 (NM_001293798.2) sequences, using BLAST (NIH).

Both strands of all PCR products were sequenced on an ABI 3730 capillary sequencer using the BigDye v3.1 cycle sequencing kit (Applied Biosystems, Inc., Norwalk, CT, USA) and the same primers as in the initial PCR. Forward and reverse sequences were aligned and edited in Sequencher v.5.0.1 (Gene Codes) and a contig of all sequences exported to eBioX v.1.5.1 (http://www.ebioinformatics.org) for final alignment using MUSCLE (Edgar 2004 (link)). A haplotype network was subsequently estimated using the TCS program (v.1.21; Clement et al. 2000 (link)) using the default 95% connection limit. In addition, annotation of the sequenced region was accomplished by analyses of the full mitochondrial genome for S. carcini (unpubl. data Podsiadlowski, L., Hecht, J., Rees D., Noever, D., Glenner, H.) using the MITOS web server (Bernt et al. 2012 (link); http://mitos.bioinf.uni-leipzig.de/).

PCR was performed on both RT- and RT+ cDNA samples with Amplitaq Gold DNA Polymerase (Thermo Fisher Scientific) or PfuUltra II Fusion High-fidelity DNA polymerase (Agilent) and primer pairs corresponding to the newly identified SVV transcripts (S5 Table). PCR amplification was performed by initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation (30 s, 95°C), primer annealing (50°C for 30 s) and subsequent primer extension (1–1.5 min at 72°C), and finally a single extension step of 10 min at 72°C. Adaptor-based and nested PCRs were performed with the same protocol on PCR reactions purified by Qiagen MinElute Reaction Clean up kit. For strand-specific RT-PCR, we first generated strand-specific cDNA using K20T primers (consisting of 20 thymidines and an “adaptor K” sequence) (S5 Table) and subsequently used transcript-specific forward primer and “adaptor K” specific reverse primer (PrK) (S5 Table). For RNA13.5–1, the strand-specific PCR was purified and followed by a semi-nested PCR with RNA13.5–1 specific forward and reverse primers. Amplicons were visualized on an agarose gel, excised from gel, purified using the Qiaquick Gel Extraction kit and subsequently sequenced with the BigDye v3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI Prism 3130 XL Genetic Analyzer.