title: GSM4150354: p4_recovery_6h_rep3; Arabidopsis thaliana; Bisulfite-Seq
library_construction_protocol: Genomic DNA was extracted using the micro C-TAB procedure. The purity of the DNA samples was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, USA), whereas the integrity was evaluated using agarose gel electrophoresis. An exact estimation of genomic DNA concentration was conducted using fluorimetry with dsDNA specific dye and QuantiFluor Single Tube Fluorometer instrument (Promega, Madison, USA). One µg of genomic DNA was fragmented in 50 µl of 0.5x Tris-EDTA buffer using Bioruptor Plus sonication device (Diagenode, Liège, Belgium) under ten cycles of 30 s ON and 90 s OFF power conditions. Then, fragmented DNA was purified with 1.8x Agencourt AMPure XP (Beckman Coulter, Brea, USA) and eluted in 40 µl of ddH2O. Whole genome bisulfite sequencing libraries were prepared using the NEXTflex Bisulfite-Seq Kit (BIOO Scientific, Austin, USA) according to the manufacturer's instructions with minor changes. Briefly, genomic DNA fragments were subjected to end repair, clean-up and adenylation. Next, barcoded adapters were ligated. The ligation products were purified and size-selected with Agencourt AMPure XP (Beckman Coulter, Brea, USA). Prepared adapter-ligated fragments were subjected to bisulfite conversion with the EZ DNA Methylation-Lightning Kit (Zymo Research, Irvine, USA) according to manufacturer's protocol, and eluted in 17 µl of M-Elution buffer. Five µl of converted DNA was amplified in 50 µl volume of PCR reaction. Amplicons were purified twice with Agencourt AMPure XP (Beckman Coulter, Brea, USA).
attributes.tag: GEO Accession
attributes.value: GSM4150354