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Sample GSM1240018 Query DataSets for GSM1240018
Status Public on Sep 30, 2014
Title BstUI Mmusculus - bisSeq 3
Sample type SRA
Source name mouse embryonic stem cells
Organism Mus musculus
Characteristics library type: natural genomic fragments
replicate type: biological replicate
cell type: mouse embryonic stem cells
genetic background: 129S6
restriction enzyme: BstUI
Treatment protocol The Recombinase-mediated Cassette Exchange (RMCE) insertion protocol was adapted from Lienert et al to scale the needs of inserting large number of fragments in paralell. Briefly, TC-1 ES cells were selected under hygromycin (250μg/ml, Roche) for 10 days. Next, 12x106 cells were electroporated (Amaxa nucleofection, Amaxa) with 75 μg of L1-library-1L plasmid and 45 μg of pIC-Cre. Negative selection with 3 μM Ganciclovir (Roche) was started 2 d after transfection and continued for 10 days. Pools of selected cells were tested for successful insertion of DNA libraries by PCR using primers recognizing the universal priming region flanking the insertion site.
Growth protocol Wild type embryonic stem cells derived from pure 129S6 background blastocysts, were cultivated on feeder cells or 0.2% gelatine coated dishes. ES cell growth medium consisted of DMEM (Invitrogen) supplemented with 15% foetal calf serum (Invitrogen), 1x non-essential amino acids (Invitrogen), 1mM L-glutamine, LIF and 0.001% beta-mercaptoethanol.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA (2μg) of ES cells carrying the libraries was bisulfite converted with the EpiTec Bisulfite Kit (QIAGEN). Libraries were amplified by PCR (AmpliTaq Gold-Invitrogen) using bisulfite compatible primers (5’-AACCTAACTATAATAAACAACC-3’; 5’-GGTATATGTATTTTTTTAGGGT-3’) annealing to the universal priming region flanking the fragments cloning site. PCR product was gel purified and fragmented by sonication (Covaris S220). The sonicated material was used to construct sequencing libraries following Illumina’s recommendations. Samples were sequenced as barcoded pools on Illumina GAII or MiSeq instruments.
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina MiSeq
Description Sub representation of the Mouse genome by size selected BstUI digest
Data processing 150bp long reads were splited in three instances (40-70-40bp). Short reads were used directly.
Bismark/ Bowtie 0.12.7 were used to align bisulfite reads against an in silico converted reference genome (C>T and G>A) and call methylation state for each CG. (mouse-mm9; Ecoli-NC_010473.1)
CGs covered by at least 10 reads were used for analysis. Strain specific SNPs were masked. Methylation was called per CG and fragment averages were derived using the previously established reference set of regions for the library. Only fragments where >50% of the CG and a minimum of 4 CGs were covered were considered in the analysis.
Genome_build: mm9/NC_010473.1
Supplementary_files_format_and_content: tab delimited fragment report : Fragment level average methylation call
Submission date Sep 25, 2013
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 GPL16417
Series (1)
GSE51170 Identification of building principles of methylation states at CG rich regions by high-throughput editing of a mammalian genome
BioSample SAMN02363967
SRA SRX360368

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

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