GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM748788 Query DataSets for GSM748788
Status Public on Dec 09, 2011
Title MouseNP_BisSeq_HiSeq
Sample type SRA
Source name ES-derived neuronal progenitor cells
Organism Mus musculus
Characteristics strain: Mixed (129-C57Bl/6)
cell type: ES-derived neuronal progenitor cells
Growth protocol Wild-type embryonic stem cells (129-C57Bl/6) were cultured and differentiated as previously described (M. Bibel, J. Richter, E. Lacroix, Y. A. Barde, Nat Protoc 2, 1034 (2007))
Extracted molecule genomic DNA
Extraction protocol The protocol was adapted from Illumina Genomic DNA Sample Preparation Guide and Paired-End Sample Preparation Guide. Briefly, One to five ug of input DNA were fragmented by sonication to 50-500 bp with a Bioruptor (Diagenode, Sparta, NJ). DNA fragments were end repaired by incubation at 20 degrees C. for 30 minutes with 400uM dNTP, 15 units of T4 DNA polymerase (NEB #M0203S), 5 units of DNA Polymerase I Lg. Frag. (Klenow) (NEB #M0210S), 50 units of T4 PNK (NEB #M0201S), 1x T4 DNA ligase buffer containing 10mM ATP (NEB). 3' ends of DNA fragments were adenylated by incubation at 37 degrees C. for 30 minutes with 200uM dATP, 1xNEB Buffer 2, 15 units Klenow Fragment (3'-> 5' exo-)(NEB # M0212L). Adapter sequences were reproduced based on Illumina adapter sequences (Oligonucleotide sequences 2006-2008 Illumina, Inc. All rights reserved). For single end sequencing: 5' P-GATXGGAAGAGXGGTTXAGXAGGAATGXXGAG and 5' AXAXTXTTTXXXTAXAXGAXGXTCTTXXGATXT, and for paired end sequencing: 5' P-GATXGGAAGAGXGGTTXAGXAGGAATGXXGAG and 5' AXAXTXTTTXXXTAXAXGAXGXTXTTXXGATXT where X is a methylated cytosine. Adapters were ordered as single stranded oligos (Microsynth AG), resupended in annealing buffer (10mM Tris pH7.5, 50mM NaCl, 1mM EDTA), annealed by heating at 95 degrees C. for 10 minutes and cooling down slowly. Annealed adapters were ligated to the DNA fragments as per manufacturer's instructions for genomic DNA library construction. Adapter-ligated DNA of 140-210 bp (for single end sequencing) or 340-410 (for paired end sequencing) was isolated by 2% agarose gel electrophoresis. Gel purified DNA was then converted with sodium bisulfite using the Imprint DNA Modification Kit (Sigma-Aldrich) as per manufacturer's instructions. One third of the bisulfite-converted, adapter-ligated DNA molecules were enriched by 7 cycles of PCR with the following reaction composition: 2.5 U of uracil-insensitive PfuTurboCx Hotstart DNA polymerase (Stratagene), 5ul 10X PfuTurbo reaction buffer, 25uM dNTPs, 0.5uM of Illumina PCR primers. The thermocycling parameters were: 95C 2 min, 98C 30 sec, then 7 cycles of 98C 15 sec, 65C 30 sec and 72C 3 min, ending with one 72C 5 min step. The reaction products were purified using the MinElute PCR purification kit (Qiagen, Valencia, CA), separated by 2% agarose gel electrophoresis to separate the library from adapter-adapter ligation products, and purified from the gel using the MinElute gel purification kit (Qiagen, Valencia, CA). Quality of the libraries and template size distribution were assessed by running an aliquot of the library on an Agilent 2100 Bioanalyzer (Agilent Technologies).
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina HiSeq 2000
Description bisulfite converted
Data processing All C nucleotides in sequence reads from bisulfite converted samples were converted in silico to T nucleotides, and the converted reads were aligned to a similarly converted genome separately to each strand using the software bowtie (version et al., Genome Biol. 2009;10(3):212) with parameters --best --strata -v 3 --norc -a. Only reads with a unique alignment in this reduced alphabet base-space were retained, and C nucleotides from the original reads and genome were reintroduced. To eliminate effects caused by polymorphisms in our experimental system, C nucleotides that overlapped known SNPs between the reference C57BL/6J and the 129S5 strains were removed from further analysis based on the SNPs identified by the Mouse Genomes Project at Sanger Institute (downloaded from Total and methylated counts for all covered CpGs in the genome were calculated as the number of alignments with either C (methylated) or T (unmethylated) and the number of alignments with C (methylated), combining the counts from the two Cs in a CpG and its reverse complement (position i on plus strand and position i+1 on minus strand).
Submission date Jun 27, 2011
Last update date May 15, 2019
Contact name Dirk Schuebeler
Organization name Friedrich Miescher Institute for Biomedical Research
Street address Maulbeerstrasse 66
City Basel
ZIP/Postal code 4058
Country Switzerland
Platform ID GPL13112
Series (2)
GSE30202 DNA binding factors shape the mouse methylome at distal regulatory regions [BIS_seq]
GSE30206 DNA binding factors shape the mouse methylome at distal regulatory regions.
SRA SRX080193
SRA SRX080194
SRA SRX080195
BioSample SAMN00631234

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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