Epitect plus dna bisulfite conversion kit

For somatic tissue, rosette leaves were pooled from 10 plants for each treatment group. For male gamete analysis, sperm and vegetative nuclei were collected from 100 plants for each treatment group. gDNA was extracted from leaf samples with the DNAeasy Plant Mini Kit (Qiagen), and from sperm and vegetative nuclei with MasterPureDNA Purification Kit (Epicentre). DNA libraries were generated using the Illumina TruSeq Nano kit (Illumina, CA, USA). DNA was sheared to 350 bp. The bisulfite treatment step using the Epitect Plus DNA Bisulfite Conversion Kit (Qiagen, Hilden, Germany) was inserted after the adaptor ligation; incubation in the thermal cycler was repeated once before clean-up. After clean-up of the bisulfite conversion reaction, library enrichment was done using Kapa Hifi Uracil+ DNA polymerase (Kapa Biosystems, MA, USA). Libraries were sequenced with 2 x 101 bp paired-end reads on an Illumina HiSeq 2000 instrument, with conventional gDNA libraries in control lanes for base calling calibration. Seven to eight libraries with different indexing adapters were pooled in one lane. For image analysis we used Illumina RTA 1.13.48.

Genomic DNA was extracted from leaves of hybrid and parent seedlings with the DNAeasy Plant Mini Kit (Qiagen). DNA was sheared to 350 bp with a Covaris™ S220 sonicator. DNA end repair, A-tailing, and adaptor ligation were performed with a TruSeq Nano Kit (Illumina) according to the manufacturer’s instructions. After adaptor ligation, the bisulfite treatment was done with an Epitect Plus DNA Bisulfite Conversion Kit (Qiagen). PCR amplification of library molecules was done using KAPA Hifi Uracil + DNA Polymerase (Kapa Biosystems). Libraries were sequenced on an Illumina HiSeq 3000 instrument with 2 × 150 bp reads.
All paired-end reads were aligned to the synthetic hybrid reference genome using Bismark (v0.15.0) [107 (link)] with Bowtie 2 aligner (v2.2.4) [108 (link)] with default parameter settings. PCR duplicates were removed after mapping. The unmethylated and methylated cytosine residue(s) in every read were identified in all sequence contexts (CG, CHG, and CHH) by the “bismark_methylation_extractor” script in Bismark. The methylation ratio of a given genomic region was calculated as the ratio between the total number of identified 5-methylcytosines and the total number of sequenced cytosines.

Rosette leaves from five 4 week old plants were pooled for each sample. Genomic DNA was extracted with the DNeasy Plant Mini Kit (Qiagen, Germany). DNA libraries were generated using the Illumina TruSeq Nano kit (Illumina, CA, USA). DNA was sheared to 350 bp. The bisulfite treatment step using the Epitect Plus DNA Bisulfite Conversion Kit (Qiagen, Germany) was inserted after the adaptor ligation; incubation in the thermal cycler was repeated once before clean-up. After clean-up of the bisulfite conversion reaction, library enrichment was done using Kapa Hifi Uracil+ DNA polymerase (Kapa Biosystems, USA). Libraries were sequenced with 2 × 150 bp paired-end reads on an HiSeq 4000 (Illumina), with conventional gDNA libraries in control lanes for base calling calibration. Sixteen to 24 libraries with different indexing adapters were pooled in each lane.