title: Methylation Study of Arabidopsis mutant Atelp2 WGS
design: Genomic DNA was extracted from ~1 g of fresh rosette leaves of 4-week-old soil-grown plants.  Approximately 2 µg of high molecular weight genomic DNA were dissolved in 125 µl TE (10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA) and transferred to 6 ´ 16 mm glass microtubes with AFA fiber & snap-caps (Covaris, Woburn, MA).  DNA samples were sheared into fragments with an average size of 300 bp in a Covaris S2 ultrasonic disruptor following the manufacturer's recommended settings.  AMPure magnetic beads (Beckman Coulter, Brea, CA) with a bead-to-sample ratio of 73:100 were used to clean up the samples prior to sequencing library construction.  Sequencing libraries were made using the TruSeq DNA Sample Preparation kits (Illumina, San Diego, CA) following the manufacturer's protocol with a few modifications.  Briefly, DNA fragments were end-repaired, adenylated, adaptor-ligated, and size-selected (250-500 bp) in a 2 % agarose gel.  The gel was stained with Invitrogen SYBR safe (Life Technologies, Carlsbad, CA) and viewed on a blue light transilluminator (Life Technologies) in order to avoid UV damage.  Libraries were quantitated in a Qubit fluorometer (Life Technologies).  The final yield was approximately 160 ng (in 25 µl Tris-HCl, pH 8.0), which was subjected to sodium bisulfite treatment using the EpiTect Bisulfite kit (Qiagen) following the manufacturer's instructions.  The resulting library was amplified using uracil-insensitive PfuTurbo Cx Hotstart DNA polymerase (Agilent Technologies, Santa Alara, CA) under the following conditions: denaturation at 98°C for 30 sec, 18 cycles (98°C for 15 sec, 60°C for 30 sec, and 72°C for 1 min), and final extension at 72°C for 5 min.  The PCR amplification products were then cleaned twice using AMPure magnetic beads with a bead-to-sample ratio of 85:100, and the resulting DNA was quantitated by the Qubit fluorometer and qPCR with the Kapa SYBR Fast qPCR reagents (Kapa Biosystems, Cambridge, MA) on an ABI7900HT real-time PCR system (Life Technologies).  The average insert size of the libraries was ~360 bp.  Libraries were then diluted to 9 pM for cluster generation on the cBOT (Illumina), and a 101 cycle multiplex single-end sequencing run for pooled barcoded libraries and a 2 ´ 101 cycle multiplex pair-end sequencing run for each library were performed on an Illumina Genetic Analyzer IIx (running SCS2.9) using an 8-lane flow cell.  Sequencing was performed in the ICBR at the University of Florida.
library_name: paired-end