title: GSM4332287: Leaves, GmNRPD1-RNAi-NT-BS-Seq-r2; Glycine max; Bisulfite-Seq
library_construction_protocol: Mature leaf tissues were quickly frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. Genomic DNA was isolated using the DNAzol ES (MRC, DN128) following the manufacturer's protocol. One microgram of genomic DNA with five nanograms of spiked unmethylated lambda DNA (Promega, D1521), was sonicated into 150-300bp fragments using a Covaris S220 Focused-ultrasonicator (Covaris) with the Target BP Peak at 200, in a 130 µl sample volume in a microTUBE AFA Fiber Snap-Cap tube. The sonicated DNA was end-repaired using the End-It DNA End-Repair Kit (Epicentre, ER0720) and purified with Agencourt AMPure XP beads (Beckman Coulter, A63881). The repaired DNA was subjected to 3'end adenylation using Klenow 3'-5' exo- (NEB, M0212S) and ligated using T4 DNA ligase (NEB, M0202L) with methylated  adapters from the TruSeq DNA LT Sample Prep Kit (Illumina, FC121-4001).  The adapter-ligated DNA was bisulfite-treated using the MethylCode Bisulfite Conversion Kit (Thermo-Fisher, MECOV50) and libraries were made following the TruSeq DNA LT Sample Prep Kit protocol. The libraries were sequenced on an Illumina HiSeq X10 using PE150 mode at Berry Genomics.
identifiers.namespace: GEO
identifiers.id: GSM4332287