|
| Status |
Public on Jan 25, 2021 |
| Title |
27_M_KO_30_RRBS |
| Sample type |
SRA |
| |
|
| Source name |
Male_KO_30_soleus muscle
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
Sex: Male
genotype: KO
timepoint: 30
tissue: soleus muscle
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
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
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
reads were preprocessed with Trim Galore v. 0.4.0, a wrapper for cutadapt v. 1.9.1, with the --rrbs flag set
trimmed reads were aligned to the mm10 genome assembly using Bismark v. 0.18.1 with default parameters
CpG methylation calls were summarized using the bismark2bedgraph script which is part of the Bismark software suite
For each CpG, observations from both strands were added together to form an overall methylation level per CpG, and only CpGs covered by eight or more reads in every sample was retained
Genome_build: mm10
Supplementary_files_format_and_content: csv file with CpGs in rows and genomic annotation along with number of methylated and unmethylated observations for each sample in columns.
|
| |
|
| Submission date |
Jun 12, 2020 |
| Last update date |
Jan 28, 2021 |
| Contact name |
Lars Roed Ingerslev |
| E-mail(s) |
ingerslev@sund.ku.dk
|
| Organization name |
Copenhagen University
|
| Department |
NNF Center for Basic Metabolic Research
|
| Lab |
Integrative Physiology
|
| Street address |
Blegdamsvej 3B
|
| City |
Copenhagen |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE152348 |
Ablation of DNA-methyltransferase 3A in skeletal muscle does not affect energy metabolism or exercise capacity [RRBS] |
| GSE152349 |
Ablation of DNA-methyltransferase 3A in skeletal muscle does not affect energy metabolism or exercise capacity |
|
| Relations |
| BioSample |
SAMN15222786 |
| SRA |
SRX8537037 |
