title: GSM3684646: zr1699_6 mutant2 Treated; Arabidopsis thaliana; Bisulfite-Seq
library_construction_protocol: DNA was extracted from 100 mg of leaves powder from 3 biological replicates using CTAB extraction protocol. We applied 500 µl CTAB and 2 µl/ml β-mercaptoethanol, and samples were incubated 1h at 60 ºC. Phases separation was done with 1 vol. of chloroform isoamyl (24:1; v:v) and for the precipitation was applied 1 vol. of isopropyl. DNA was resuspended in H2O DEPC. LiCl 2M was used for a first step of DNA purification, to remove RNA. DNA was precipitated with 0.1 vol. of AcNa 3M and 2 vol. of etanol, and resuspended in H2O DEPC. 1 μg of DNA of each sample was resuspended in 50 μl of TE buffer to send them to Zymo Research (Irvine, CA) for the next steps in DNA methylation analysis (library construction protocol, sequencing run, data processing and data analysis). There, samples were treated with RNAse (RNA removal) followed by clean-up using their genomic DNA Clean & Concentrator kit (Cat. No. D4010). Then, DNA from each sample was quantified and applied a QC check using the Qubit instrument according to the manufacturer's guidelines (Qubit 3.0 Fluorometer; Life Technologies – Carlsbad, CA, USA). Methyl-MaxiSeq® libraries were prepared from 80 ng of genomic DNA digested with 2 units of Zymo Research's (ZR) dsDNA Shearase™ Plus (Cat#: E2018-50). The fragments produced were end-blunted and 3'-terminal-A extended, then purified using the Zymo Research (ZR) DNA Clean & Concentrator™ – 5 kit (Cat#: D4003). The A-tailed fragments were ligated to pre-annealed adapters containing 5'-methyl-cytosine instead of cytosine and adapter-ligated fragments were filled-in. Bisulfite treatment of the fragments was carried out using the EZ DNA Methylation – Lightning kit (ZR, Cat#: D5030). PCR was performed with Illumina TruSeq indices and the size and concentration of the fragments (library insert size) were confirmed using the D1000 screen tape on the Agilent 2200 TapeStation (according to manufacturer's instructions (Santa Clara, CA, USA)), then sequenced on an Illumina HiSeq instrument.
identifiers.namespace: GEO
identifiers.id: GSM3684646