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| Status |
Public on Aug 22, 2019 |
| Title |
O8_TA_Hi_7 [scBS-seq] |
| Sample type |
SRA |
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| Source name |
Tibialis anterior muscles
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| Organism |
Mus musculus |
| Characteristics |
strain: MGI: 5308730
cell type: Muscle stem cells
age: 24 months
mouse id: O8
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| Treatment protocol |
Mice were sacrificed by cervical dislocation. Tibialis anterior muscles were dissected and placed into cold DMEM (ThermoFisher, 31966). Muscles were then chopped and put into a 15 ml Falcon tube containing 10 ml of DMEM, 0.08% collagenase D (Sigma, 11 088 882 001), 0.1% trypsin (ThermoFisher, 15090), 10 µg/ml DNaseI (Sigma, 11284932) at 37°C under gentle agitation for 25 min. Digests were allowed to stand for 5 min at room temperature and the supernatants were collected on 5 ml of foetal bovine serum (FBS; Gibco) on ice. The digestion was repeated 3 times until complete digestion of the muscle. The supernatants were filtered through a 70-µm cell strainer (Miltenyi, 130-098-462). Cells were spun for 15 min at 515g at 4°C and the pellets were resuspended in 1 ml freezing medium (10% DMSO (Sigma, D2438) in foetal calf serum (FCS, Invitrogen)) for long term storage in liquid nitrogen. Before FACS isolation, samples were thawed in 50 ml of cold DMEM, spun for 15 min at 515g at 4°C. Pellets were resuspended in 300 µl of DMEM 2% FCS 1 µg/mL propidium iodide (Calbiochem, 537060) and filtered through a 40-µm cell strainer (BD Falcon, 352235). Viable cells were isolated based on size, granulosity and GFP expression level (top 10% nGFPHi cells) using a MoFlo Astrios cell sorter (Beckmann Coulter). Single cells were collected in 2.5 µL cold RLT Plus buffer (Qiagen, 1053393) containing 1U/µL RNase inhibitor (Ambion, AM2694) in 96 well-plates (LoBind Eppendorf, 0030129504), flash-frozen on dry ice and stored at -80°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
We prepared scM&T-seq libraries by isolating mRNA on magnetic beads and separating from the single-cell lysate prior to reverse transcription and amplification using Smartseq2 but with 25 PCR cycles.
scM&T-seq (C. Angermueller et al., 2016)
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Libraries were sequenced on the Illumina HiSeq platform using the default RTA analysis software.
BS-Seq sequences were trimmed with Trim Galore (v0.5.0; Cutadapt v1.15) in single-end mode with the options --clip_r1 6. Trimmed sequences were mapped to the mouse GRCm38 genome in single-end, --non_direcional mode using Bismark (Krueger and Andrews, 2011) (v0.20.0); CpG methylation calls were extracted and analysed using SeqMonk (www.bioinformatics.babraham.ac.uk/projects/seqmonk/).
Genome_build: GRCm38
Supplementary_files_format_and_content: *.cov: Bismark coverage files: The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>
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| Submission date |
Oct 17, 2018 |
| Last update date |
Aug 22, 2019 |
| Contact name |
Felix Krueger |
| E-mail(s) |
fkrueger@altoslabs.com
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| Organization name |
Altos Labs
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| Department |
Bioinformatics
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| Street address |
Granta Park
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| City |
Cambridge |
| ZIP/Postal code |
CB21 6GP |
| Country |
United Kingdom |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE121436 |
Ageing affects DNA methylation and transcriptional cell-to-cell variability in muscle stem cells [scBS-seq] |
| GSE121437 |
Ageing affects DNA methylation and transcriptional cell-to-cell variability in muscle stem cells |
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| Relations |
| BioSample |
SAMN10255107 |
| SRA |
SRX4901277 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3436349_O8_TA_Hi_7.cov.gz |
22.8 Kb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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