title: GSM4586658: 150ng-6PCR-EM_replicate1; Arabidopsis thaliana; Bisulfite-Seq
library_construction_protocol: Genomic DNA from Arabidopsis flowers and leaves was extracted using DNeasy Plant Mini Kit (Qiagen). Concentration of the DNA was measured by Qubit dsDNA Broad-Range Assay kit (ThermoFisher). 50 μl aliquots containing 25, 50, 150, and 400 ng DNA were sheared by an S2 Focused-ultrasonicator (Covaris) to ~200 bp in average size using these parameters: intensity 5, duty cycle 10%, cycles per burst 200, and treatment time 120 sec. EM-Seq libraries were prepared from sheared DNA using Enzymatic Methyl-seq kit following manufacturer instructions (New England BioLabs). For each input amount, three PCR conditions were used: 6, 12, and 18 cycles. Sheared DNA was end-repaired and ligated with TruSeq DNA single adapters (Illumina) using Kapa DNA HyperPrep kit (Roche). Adapter-ligated DNA was converted by EpiTect Bisulfite Kit (Qiagen). And converted DNA was amplified by MyTaq polymerase (Bioline) for 6, 12, or 18 cycles.