NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM3436276 Query DataSets for GSM3436276
Status Public on Aug 22, 2019
Title O1_TA_Hi_24 [scBS-seq]
Sample type SRA
 
Source name Tibialis anterior muscles
Organism Mus musculus
Characteristics strain: MGI: 5308730
cell type: Muscle stem cells
age: 24 months
mouse id: O1
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.
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)
 
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina HiSeq 2500
 
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>
 
Submission date Oct 17, 2018
Last update date Aug 22, 2019
Contact name Felix Krueger
E-mail(s) fkrueger@altoslabs.com
Organization name Altos Labs
Department Bioinformatics
Street address Granta Park
City Cambridge
ZIP/Postal code CB21 6GP
Country United Kingdom
 
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
Relations
BioSample SAMN10254925
SRA SRX4901204

Supplementary file Size Download File type/resource
GSM3436276_O1_TA_Hi_24.cov.gz 218.4 Kb (ftp)(http) COV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap