GSM2101447: Root tip (Col-0) MethylC-seq; Arabidopsis thaliana; Bisulfite-Seq
Fluorescent Activated Cell Sorting (FACS) was performed using cell specific GFP lines. Sorted cells were collected directly into specific lysis buffers that were compatible with downstream applications. Cells used for bisulfite sequencing, mRNA-seq, smRNA-seq were lysed in Buffer AP1 (Qiagen), Buffer RLT (Qiagen), Trizol (Invitrogen). All samples were immediately stored at -80 °C until gDNA and RNA was extracted using DNeasy Plant mini kit (Qiagen) and RNeasy Plant mini kit (Qiagen) or Trizol, respectively. Approximately one to three micrograms of genomic DNA was sonicated to ~100 bp using the Covaris S2 System using the following parameters: cycle number = 6, duty cycle = 20%, intensity = 5, cycles/burst = 200 and time = 60 seconds. Sonicated DNA was purified using Qiagen DNeasy minielute columns (Qiagen). Each sequencing library was constructed similar to genomic DNA libraries except the ligation was performed with methylated adapters provided by Illumina. Ligation products were purified with AMPure XP beads (Beckman) at a ratio of 1.8X of beads to sample. Up to 450 ng of ligated DNA was bisulfite treated using the MethylCode Kit (Invitrogen, Carlsbad, CA) following the manufacturer’s guidelines and then PCR amplified using Pfu Cx Turbo (Agilent, Santa Clara, CA) or KAPA HIFI Uracil+ (KAPA Biosystems, Wilmington, MA) using the following PCR conditions (2 minutes at 95C, 4 cycles of 15 seconds at 98C, 30 seconds at 60C, 4 minutes at 72C and 10 minutes at 72C).
GEO
GSM2101447