GSM5049375: 40_F_WT_10_RRBS; Mus musculus; Bisulfite-Seq
A single soleus muscle or ~30 mg of powdered quadriceps was homogenized in 400 µl of RLT buffer (Qiagen) with β-mercaptoethanol using steal beads and subjected to 2 x 30 sec at 30 Hz of disruption using the TissueLyser II (Qiagen). RNA and DNA were simultaneously isolated from 350 µl of lysate using the RNA/DNA/miRNA AllPrep kit (Qiagen) as per instructions. Primary muscle cells were lysed directly in RLT buffer and RNA extracted using the RNeasy kit (Qiagen). RNA was eluted in 45 µl of RNAse free water and DNA in 60 µl of elution buffer (Qiagen). Reduced Representation Bisulfite Sequencing (RRBS) was performed as described (Laker et al., 2017). Briefly, DNA samples were incubated with MpsI enzyme (NEB) in order to fragment DNA at CCGG positions to enrich for CpG regions. Bisulfite conversion was performed using the EZ DNA methylation Kit (Zymo Research) according to the manufacturer's instructions. The DNA was then PCR amplified and ligated to TruSeq (Illumina) sequencing adapters. Libraries were subjected to 75-bp single-end sequencing on the NextSeq 550 (Illumina). On average 18.3 million reads/sample were aligned