Low range ultra agarose

The RNA-Seq transcriptome libraries were prepared using a TruSeq RNA sample preparation kit from Illumina (San Diego, CA, USA) with 5 μg total RNA. mRNA was then isolated according to the polyA selection method using oligo(dT) beads and fragmented in fragmentation buffer. Next, double-stranded cDNA was then synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) with random hexamer primers (Illumina). The synthesized cDNA was subsequently subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina's library construction protocol. Libraries were size-selected for cDNA target fragments of 200–300 bp on 2% Low Range Ultra Agarose (Bio-Rad, Hercules, CA, USA) followed by PCR amplification using Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA) for 15 PCR cycles. After quantification using a TBS380 Fluorometer (Turner Biosystems Inc., Sunnyvale, CA, USA), a paired-end RNA-Seq library was sequenced with an Illumina HiSeq 4000 instrument (2 × 150 bp read length). The sequencing data were deposited in the NCBI/SRA database (Bioproject: PRJNA422178; BioSample: SAMN08166800; Sequence Read Archive Database under accession number SRP126885).

RNA was extracted using the Qiagen RNeasy Plant Mini Kit (Qiagen), with an additional sonication step after addition of buffer RLT. RNA integrity was checked using the Agilent BioAnalyzer 2100 (Agilent). Approximately 2 µg of total RNA was used for mRNA-Seq library construction according to the manufacturer’s recommendations (Illumina).
We used the multiplexing sequencing adapters provided in the Multiplexing Sample Preparation Oligo Kit (Illumina). Size selection of the library was performed on a 2% agarose gel (Low Range Ultra Agarose, Biorad 161-3107). The denatured library was diluted to a final concentration of 6 pM and loaded on a paired-end read flow cell (TruSeq v5 kit, Illumina). To minimize lane effects the samples were multiplexed. Each sample was sequenced in duplicate in 2 different lanes (4 lanes total with 8 MID tags per lane). After cluster generation, the multiplexed library was sequenced on an Illumina Genome Analyzer IIx (36 cycles, paired end).

Verification of the karyotype was conducted using a pulsed-field gel electrophoresis system (DRII; Bio-Rad, Munich, Germany). Plugs containing intact chromosomes were produced from a Z. tritici cell suspension by centrifugation (3,500 × g, 10 min) and resuspension of the cell pellet in 1 ml double-distilled water (ddH₂O). Whole cells were embedded in 1 ml 2.2% Low-Range Ultra agarose (Bio-Rad, Munich, Germany)–0.5× Tris-borate-EDTA (TBE) buffer and incubated twice for 24 h each time at 55°C in 5 ml lysis buffer (1.5 mg/ml proteinase K, 1% SDS, 0.45 M EDTA, pH 8.0). Small chromosomes were separated in 1.2% pulsed-field agarose (Bio-Rad, Munich, Germany)–0.5× TBE buffer and were processed for 48 h at 14°C using an angle of 120°, 5 V/cm, and a switching time of 50 to 150 s. Midsize chromosomes were separated in 1% pulsed-field agarose–1× TBE buffer and were processed for 72 h at 14°C using an angle of 106°, 3 V/cm, and a switching time of 250 to 1,000 s. Large chromosomes were separated in 0.8% pulsed-field agarose–1× TRIS-acetate-EDTA (TAE) buffer and were processed for 92 h at 13°C using an angle of 106°, 2 V/cm, and a switching time of 1,000 to 2,000 s. Gels were stained for 30 min in a 0.5 µg/ml ethidium bromide solution followed by 10 min of destaining in H₂O.