GSM2650209: clsy2-2_MethylC-seq; Arabidopsis thaliana; Bisulfite-Seq
0.1g of un-opened flower buds (stage 12 and younger) were collected from the same individual plants as used for the smRNA-seq analyses and genomic DNA was isolated using the DNeasy Plant Mini Kit (Qiagen, Cat# 69104). 2.0μg purified genomic DNA then was sonicated using the Covaris S2 Sonicator and fragments that smaller than 500bp were retained using Sera-Mag Magnetic SpeedBeads (Thermo scientific, Cat# 65152105050250) for MethylC-seq libraries. These DNA ends were repaired using the End-It DNA End-Repair Kit (Epicentre, Cat# ER81050) and purified by Sera-Mag Magnetic SpeedBeads. The end-repaired DNA was adenylated at 3’ end using the polymerase activity of Klenow Fragment (3’à5’ exo, NEB, Cat# M0212) and followed by purification of Sera-Mag Magnetic SpeedBeads. These DNA were then ligated to the methylated adapter in NEXTflex Biosulfite-Seq Barcode Kit (BIOO Scientific, Cat# 511911) using T4 DNA ligase (NEB, Cat# M0202). The ligated DNA was performed the C-to-T bisulfite conversion using MethylCode Bisulfite Conversion Kit (Life Tech Cat# MECOV50). The resulting DNA was amplified for 4 cycles, purified, pooled and sequenced with SE50 using HiSeq 2500.
GEO Accession
GSM2650209