|
| Status |
Public on Sep 30, 2014 |
| Title |
NarI Mmusculus - bisSeq 2 |
| Sample type |
SRA |
| |
|
| Source name |
mouse embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
library type: natural genomic fragments
replicate type: technical replicate
cell type: mouse embryonic stem cells
genetic background: 129S6
restriction enzyme: NarI
|
| 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.
http://www.illumina.com/products/chip-seq_dna_sample_prep_kit.ilmn
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| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Sub representation of the Mouse genome by size selected NarI digest
mouse_NarI_summary.tab
|
| 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 |
|
| Relations |
| BioSample |
SAMN02363962 |
| SRA |
SRX360362 |
