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| Status |
Public on Apr 08, 2014 |
| Title |
MJB137_RR_bisulphite_ESC_mouse_R2 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
rrbs_enzyme: MspI
bisulphite treatment: bisulphite
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| Treatment protocol |
no treatment applied
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| Growth protocol |
Mouse embryonic stem cells, derived in-house from Black6/Castaneous mice, were cultured on gelatine-coated plates in a DMEM-KO medium (Invitrogen) supplemented with 10% FCS, 100 µM non-essential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, 50 µg/ml penicillin/streptomycin (all GIBCO), 100 µM 2-mercaptoethanol (Sigma), 1000 U/ml mouse Leukemia Inhibitory Factor (Chemicon) and 2i (MEK inhibitor PD0325901 (Axon) and GSK-3 inhibitor CT99021 (Axon)).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were resuspended in lysis buffer (100 mM Tris, pH 5.5, 5 mM EDTA, 200 mM NaCl, 0.2% SDS), supplemented with 400 µg/ml proteinase K (Invitrogen) and 200 µg/ml RNase A (Qiagen), and were incubated at 55°C overnight. DNA was extracted using phenol-chloroform and Phase Lock Gel Light tubes (5 Prime), was precipitated in 70% ethanol and resuspended in pure water.
Samples of genomic DNA (12 x 1 µg) were digested with MspI (1 µL) in a digestion (30 µL total volume) overnight at 37 °C. To terminate the digestions, EDTA (3 µL, 0.5 M) was added. The DNA was subsequently worked up using the GeneJET PCR kit and eluted in EB (60 µL). The internal control DNA (1 µL, 0.6 ng/µL) was then added to all samples. All twelve MspI digested samples were subjected to an Illumina library preparation with the Illumina TruSeq kit using a 1/10 dilution of adapters. For the last AMPureXP purification from the TruSeq protocol the DNA was left to dry for 30 mins and eluted in water.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
reduced representation (MspI treatment) of bisulphite treatment for mouse ESC, replicate 2
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| Data processing |
fastq file processed using trim_galore (version 0.2.6) and aligned using bismark (version 0.10.0).
Modifications at each site were then counted using the command mpileup from samtools (version 0.1.18).
The previous steps have been wrapped in the program bsExpress (http://code.google.com/p/oxbs-sequencing-qc/wiki/bsExpressDoc).
Genome_build: mm9
Supplementary_files_format_and_content: Processed data files are in BedGraph Track Format (http://genome.ucsc.edu/goldenPath/help/bedgraph.html), extended with additional columns. Columns are tab-separated and they are, respectively: chromosome, start, end, modification percentage, modification count, total count, strand.
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| Submission date |
Apr 07, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Giovanni Marsico |
| E-mail(s) |
persego@gmail.com
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| Organization name |
CRUK Cambridge Institute
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| Street address |
Robinson Way
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0RE |
| Country |
United Kingdom |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE56572 |
Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution |
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| Relations |
| BioSample |
SAMN02721155 |
| SRA |
SRX512523 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1364070_MJB137.SLX-6689.A025.C28FTACXX_full_wdups.mcall.bedGraph.gz |
98.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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