GSM1575307: YJ suvh1 Bsseq rep2; Arabidopsis thaliana; Bisulfite-Seq
Genomic DNA was extracted using the DNeasy Plant Mini Kit. To generate the whole-genome bisulfite sequencing (BS-seq) libraries, genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen). One microgram of genomic DNA was sonicated into fragments 100 to 300 bp in length using a Diagenode Bioruptor. The sonicated DNA fragments were purified using the PureLink PCR Purification Kit (Invitrogen). End repair was performed using the End-It™ DNA End-Repair Kit (Epicenter) followed by purification of the Agencourt AMPure XP-PCR Purification system. 3’-end adenylation was performed at 37°C for 30 min using dATP and Klenow Fragment (3’"5’ exo-) (New England Biolabs), followed by purification using the Agencourt AMPure XP-PCR Purification system. The purified DNA was ligated with methylated adapters from the TruSeq DNA Sample Preparation Kit (Illumina). The ligation products were purified with AMPure XP beads twice. Less than 400 ng ligated product was used for bisulfite conversion using the MethylCode Kit (Invitrogen) according to the manufacturer’s guidelines, except for the addition of 12 μg carrier RNA (Qiagen) to the conversion product before column purification. The final conversion product was amplified using Pfu Cx Turbo (Agilent) under the following PCR conditions: 2 min at 95°C; 9 cycles of 15 s at 98°C, 30 s at 60°C and 4 min at 72°C; and 10 min at 72°C. The PCR product was purified using AMPure XP beads prior to a 101-cycle sequencing run (single end) on an Illumina HiSeq 2000.
GEO
GSM1575307