alias: E-MTAB-4626:control_1_B
title: Illumina HiSeq 2500 paired end sequencing; Oryza sativa ssp. indica Illumina libraries of the method plant-RRBS used for methylome profiling
design: Oryza sativa ssp. indica Illumina libraries of the method plant-RRBS used for methylome profiling
library_construction_protocol: The sample material for the molecular analysis was the 4th leaf of individual plants per each of 5 biological repeats / line, which belongs to the 4th and the 5th selfing of the EUE selection pedigree.  LR2 and the control line 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 μmol m-2 sec-1, the relative humidity was kept at 71 %).  Isolation of genomic DNA was performed from an optimal mass of about 60 mg leaf material to obtain high quality and quantity of genomic DNA, using the Wizard Genomic DNA Purification Kit (Promega) according to manufacturer's instructions, from five individual plants (biological repeats) per epiline LR2 and per control line. Genomic DNA isolates were examined for high quality and sufficient quantity by NanoDrop spectrophotometry (concentration, A260/A280 of approximately 1.8 or above) and gel electrophoresis (high molecular weight, integrity, purity). Plant-RRBS libraries for Illumina sequencing were constructed by Alpha Biolaboratory, Inc. Saratoga, CA according to Hsieh (2015) and Pignatta et al., (2015)  with modifications. Approx. 300 ng of digested genomic DNA was purified, end repaired, and ligated to custom-synthesized methylated multiplex adapters (Eurofins MWG Operon, Huntsville, AL) according to the manufacturer's (Illumina, San Diego, CA) instructions. To ensure recovery of shorter plant-RRBS library inserts, the solid-phase reversible immobilization (SPRI) method with 1.8 x (v/v) AMPure XP beads (Beckman Coulter, Brea, CA) was used for cleanup steps throughout library construction procedure. Adaptor-ligated libraries were subjected to one round of sodium bisulfite conversion with the EZ DNA Methylation-Lightning Kit (Zymo Research Corporation, Irvine, CA) as outlined in the manufacturer's instructions. Five to ten nanogram of bisulfite-converted libraries was PCR amplified with the following condition: 2.5 U of ExTaq DNA polymerase (Takara), 5 ul of 10 x Extaq reaction buffer, 25 mM dNTPs, 1 ul of index Primers (10 uM) in 50 uL reaction. The thermocyling was as follows: 95 °C for 3 min and then 12 cycles each of 95 °C for 30 s, 65 °C for 30 s, and 72 °C for 60 s. The enriched libraries were purified twice with 0.8x (v/v) AMPure XP beads to remove any adapter dimers. The library quality was assessed by randomly sub-cloning and sequencing 20 – 30 colonies to evaluate for proper library construction, bisulfite conversion, and the presence of correct indexes. Final libraries were evaluated by 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and qPCR for library size distribution and quantification.
identifiers.namespace: Department of Plant Biotechnology and Bioinformatics, Ghent University, Gent, Belgium Department of Plant Systems Biology,VIB, Gent, Belgium
identifiers.id: E-MTAB-4626:control_1_B