 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 09, 2011 |
| Title |
MouseNP_BisSeq_GAIIx |
| 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 Genome Analyzer IIx |
| |
|
| 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 0.10.0.1)(Langmead 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 ftp://ftp-mouse.sanger.ac.uk/REL-1003/SNPs/20100301-all-snps.tab.gz). 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 |
GPL11002 |
| 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. |
|
| Relations |
| SRA |
SRX080196 |
| BioSample |
SAMN00631235 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
|
|
|
|
 |
