Illumina HiSeq 2500 sequencing; Illumina whole-genome bisulfite sequencing of Oryza sativa ssp. indica inbred control and epilines
Illumina whole-genome bisulfite sequencing of Oryza sativa ssp. indica inbred control and epilines
The sample material was 12 pooled fourth leaves of individual plants of to the 4th selfing of the EUE selection pedigree.   Plants were grown in soil in a growth chamber at 26 C / 21 C (day / night) for 24 days with a 16 h light/8 h dark regime (light intensity was 300 mol m-2 sec-1, the relative humidity was kept at 71%)   DNA isolation was performed using the CTAB (cetyltrimethyl/ammonium bromide) standard protocol For WGBS, paired-end bisulfite sequencing libraries were constructed by Alpha Biolaboratory, Inc. Saratoga, CA as described previously (Ibarra et al., 2012) with modifications. About 300 ng of genomic DNA was fragmented by sonication, end repaired and ligated to custom-synthesized methylated adapters (Eurofins MWG Operon, Huntsville, AL)  according to the manufacturer's (Illumina, San Diego, CA) instructions for gDNA library construction. Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (Qiagen, Hilden, Germany) as outlined in the manufacturer's instructions. One quarter of the bisulfite-converted libraries was PCR amplified using the following conditions: 2.5 U of ExTaq DNA polymerase (Takara Bio), 5 µL of 10 x Extaq reaction buffer, 25 µM dNTPs, 1 µL primer 1.1, 1 µl primer 2.1 in a 50 µL reaction. The thermocyling conditions were as follows: 95°C for 3 min, then 12-14 cycles of 95°C for 30 s, 65°C for 30 s and 72°C for 60 s. The enriched libraries were purified twice with solid phase reversible immobilization (SPRI) method using 0.8 x v/v AM-Pure beads (Beckman Coulter, Brea, CA) prior to quantification with a Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Experimental Factor: genotype
LR1