title: GSM4890228: 20m-3; Rattus norvegicus; Bisulfite-Seq
library_construction_protocol: 500ul of whole blood was collected via retro-orbital bleedings into heparinized tubes, spun, and the plasma removed. Buffy coat and red blood cells were frozen at -80 degrees C until DNA extraction. Following proteinase K and RNAse A treatments, DNA was isolated from rat blood cells using QIAmp DNA Mini Kit following manufacturer's instructions using a QIAcube automated device. DNA was eluted from columns in 200 µL of AE buffer, concentrated in 20 µL 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA using Genomic DNA Clean & Concentrator-10, and quantified using a Qubit 2.0. 100ng of DNA isolated from whole blood was digested with MspI restriction enzyme (NEB, Ipswich, MA, USA), carried out end-repair/adenylation (NEB) and ligation with TruSeq barcoded adapters (Illumina, San Diego, CA, USA). DNA fragments of size range 200-300bp were selected with SPRI magnetic beads (ABM, Richmond, BC, CA), followed by bisulfite treatment (Millipore, Billerica, MA, USA), and PCR amplification (Bioline, Taunton, MA, USA). Libraries were sequenced by multiplexing 8 libraries per lane on the Illumina HiSeq2500 sequencer, with 100bp single end reads.