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Sample GSM821398 Query DataSets for GSM821398
Status Public on Mar 27, 2012
Title E14_high.MethylCap
Sample type SRA
Source name Embryonic stem cells
Organism Mus musculus
Characteristics origin: Embryonic stem cells
technique: MethylCap-sequencing
binding protein: His6-GST-MBD
binding protein vendor: Diagenode
Growth protocol HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (all Gibco/Invitrogen) at 37 °C in 5% CO2 atmosphere.
ES cells were cultured in normal cell culture containing ES-grade serum supplemented with LIF. These cells were grown on gelatin-coated plates
Extracted molecule genomic DNA
Extraction protocol MethylCap (Capturing of methylated DNA) was done using in automated procedure using a SX-8G/IP-StarTM robot (Diagenode). 2 ug of His6-GST-MBD (Diagenode) was incubated with 1 ug of fragmented DNA in binding buffer (BB, 20 mM Tris-HCl pH 8.5, 0.1% Triton X-100) with 200 mM NaCl in a final volume of 200 ul. This solution is mixed at 4 C for 2 h. Magnetic GST-beads were prepared by washing 35 µl of a well-mixed beads suspension (Promega) with 200 µl of binding buffer + 200 mM NaCl at 4 C. The suspension was mixed and a magnet was used to collect the beads. After removal of the supernatant the procedure is repeated once. After the second wash the supernatant is removed, but the beads are not allowed to get dry. The GST-MBD solution is added to the washed beads and this suspension is mixed for another hour at 4C. After removal of the supernatant (the 'flow-through') the beads-GST-MBD-DNA complexes are washed/eluted. 200 µl of binding buffer with increasing concentrations of NaCl is added and the suspension is rotated for 10 min at 4 C. Beads are captured on the magnet, and the supernatant is collected. Three different eluates were collected (LOW, MEDIUM, HIGH), corresponding 500 mM, 600 mM and 800 mM NaCl. For MethylCap-BS-seq, only HIGH eluates were used.
Library strategy MBD-Seq
Library source genomic
Library selection MBD2 protein methyl-CpG binding domain
Instrument model Illumina Genome Analyzer IIx
Description 1 to 30 ng was used for library preparation using the ChIP-seq DNA sample Prep Kit (Illumina, cat# IP-102–1001) using the recommended protocol. After ligation of adapters, a 300 bp band is excised using the E-Gel system (Invitrogen), which correspond to a 234 bp fragment of genomic DNA. PCR enrichment of adapter-ligated DNA was done using 14 cycles of PCR.
Data processing Cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina Genome Analyzer IIx (GAIIx) platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Sequences were aligned to the mouse (mm9) reference genome using the Illumina Analysis Pipeline allowing, one mismatch. Only the tags uniquely aligning to the genome were considered for further analysis. Further analysis was performed using the 36 bp sequence reads. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing the data in the UCSC Genome Browser. All sequence analyses were conducted based on the Mus Musculus mm9 genome assembly.
Submission date Oct 24, 2011
Last update date May 15, 2019
Contact name Arjen Brinkman
Organization name Radboud University, Nijmegen Center for Molecular Life Sciences
Department Molecular Biology
Lab Stunnenberg
Street address NCMLS #274, Geert Grooteplein Zuid 30
City Nijmegen
ZIP/Postal code 6525 GA
Country Netherlands
Platform ID GPL11002
Series (1)
GSE28254 Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk
SRA SRX105269
BioSample SAMN00752492

Supplementary file Size Download File type/resource
GSM821398_E14_high.MethylCap.reads.bed.gz 64.1 Mb (ftp)(http) BED
GSM821398_E14_high.MethylCap.reads.wig.gz 14.5 Mb (ftp)(http) WIG
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Raw data are available in SRA
Processed data provided as supplementary file

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