GSM5006073: BS07 post, Obese_Muscle; Homo sapiens; Bisulfite-Seq
Homogenization of the muscle biopsy was performed in 1X PBS with the Bullet Blender (Integrated Scientific Solutions). DNA was isolated using QIAamp DNA mini kit (Qiagen). Briefly, DNA was digested with Msp1 (New England Biolabs) and purified using QIAquick Nucleotide Removal Kit (Qiagen).  End-repair A tailing was performed (New England Biolabs) and TruSeq methylated indexed adaptors (Illumina) were ligated with T4 DNA ligase (New England Biolabs). Size selection was performed with Agencourt AMPure XP beads (Beckman Coulter). Bisulfite conversion was performed using EZ-DNA Methylation Kit (Zymo Research) as recommended by the manufacturer with the exception that incubation was performed using 55 cycles of 95 ̊C for 30 seconds and 50°C for 15 minutes.  Following bisulfite treatment, the DNA was purified as directed and amplified using Pfu Turbo C Hotstart DNA Polymerase (Agilent Technologies). The final libraries from RRBS were prepared for sequencing as per the manufacturer's instructions in the Illumina cBot and HiSeq Paired end cluster kit version 3. The flow cell was then loaded into the Illumina cBot for generation of cluster densities. After cluster generation, the flow cells were sequenced as 51 x 2 paired end reads using Illumina HiSeq 2000 with TruSeq SBS sequencing kit version 3.