End it kit

DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, D) according to the manufacturer’s protocol from segregant plant pools, and quantified using the Qubit dsDNA BR assay kit. Illumina sequencing library preparation and bisulfite conversion was conducted as described in Kawakatsu [55 (link)]. Briefly, DNA was End Repaired using the End-It kit (Epicentre, Madison, WI), A-tailed using dA-Tailing buffer and 3μL Klenow (3’ to 5’ exo minus) (NEB, Ipswich, MA) and Truseq Indexed Adapters (Illumina, San Diego, CA) were ligated overnight. Bead purified DNA was quantified using the Qubit dsDNA BR assay kit, and stored at -20°C. At least 450ng of adapter ligated DNA was taken into bisulfite conversion which was performed according to the protocol provided with the MethylCode Bisulfite Conversion kit (Thermo Fisher, Waltham, MA). Cleaned, converted single stranded DNA was amplified by PCR using the KAPA U+ 2x Readymix (Roche Holding AG, Basel, CH): 2 min at 95°C, 30 sec at 98°C [15 sec at 98°C, 30 sec at 60°C, and 4 min at 72°C] x 9, 10 min at 72°C, hold at 4°C. After amplification, the DNA was bead purified, the concentration was assessed using the Qubit dsDNA BR assay kit, and the samples were stored at -20°C. WGBS library was sequenced as part of a large multiplexed pool paired-end 150 bp on an Illumina HiSeq 4000.

Libraries were generated from ∼1.5 μg genomic DNA using the SureSelect XT Library Preparation Kit (Agilent Technologies, Santa Clara, CA, USA). DNA was fragmented with an LE220 Focus Ultra-sonicator (Covaris, Woburn, MA, USA), end repaired using the End-It kit (Epicentre, Madison, WI, USA), adenylated, ligated to sequencing adapters (Illumina, San Diego, CA, USA), and amplified by PCR. Exome libraries were captured with the SureSelect XT v4 51 Mb capture probe set (Agilent Technologies), enriched by PCR, and quantified using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA), 2100 BioAnalyzer (Agilent Technologies), and Qubit Fluorometer (Thermo Fisher Scientific). Sequencing was done on a HiSeq2500 (Illumina) using 2 × 125 bp cycles.

The method used to clone specific TRFs through enrichment of terminal sequences has been described in detail elsewhere (74 (link)). Briefly, polysaccharide-depleted, genomic DNA samples (∼2 μg) were end-repaired using the End-it kit from Epicentre Technologies (Madison, WI, USA). The enzymes were inactivated and removed using PCI (2×) and CI (1×) extraction and the DNA was ethanol-precipitated and rinsed in 70% ethanol. The DNA was then dissolved in 1× restriction enzyme buffer and digested with PstI for 4 h before being fractionated by gel electrophoresis. Gel slices containing DNA molecules in size ranges spanning the target fragments were excised and the DNA was extracted using the QIAquick kit (Qiagen, Valencia, CA, USA) and eluted in 25 μl elution buffer. The purified fragments (3.5 μl, ∼100 ng) were ligated to PstI + EcoRV-digested, CIP-treated pBlueScript KS II⁺ (∼100 ng) in a reaction volume of 5 μl. One microliter of the reaction mix was used to transform ultra-competent EPI300 cells (Epicentre). Telomere-positive clones were identified by colony blotting.