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| Status |
Public on Jan 14, 2019 |
| Title |
MethylC-seq_Ascl1_22d_B |
| Sample type |
SRA |
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| Source name |
MethylC-seq_Ascl1_22d
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| Organism |
Mus musculus |
| Characteristics |
cell type: Direct reprogramming from fibroblast to neuron induced with Ascl1 for 22 days
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| Growth protocol |
Homozygous TauEGFP knock-in mice (Tucker et al., 2001) were bred with C57BL/6 mice (Jackson Labs) to generate heterozygous embryos. Only the limbs were isolated from E13.5 embryos, chopped into small pieces, trypsinized and plated in MEF media, containing 10% cosmic calf serum (Hyclone), 0.008% Beta mercaptoethanol (Sigma), MEM non-essential amino acids, Sodium Pyruvate, and Penicillin/Streptomycin (Pen/Strep) (all from Invitrogen), to derive fibroblast cultures (called TauEGFP MEFs). For homozygous Dnmt3a/3bfl/fl MEFs, Dnmt3afl/fl and Dnmt3bfl/fl mice on a C57BL/6 background were crossed to generate Dnmt3a/3bfl/fl mice15–17 . Whole body MEFs were harvested from E13.5 embryos by removing the head, spinal cord and red organs, minced into small pieces, trypsinized and plated in MEF media on gelatinized flasks to derive fibroblast cultures. Both MEFs were expanded for three passages prior to experiments. Lentivirus was produced, and TauEGFP MEFs were co-infected with reverse tetracyclin transactivator (rtTA) and doxycycline (dox) inducible TetO-Ascl1 alone or with TetO-Brn2 and TetO-Mytl1 as previously described19. Dox was added with fresh MEF media 16-20hrs post lentiviral infection, and cultures were switched to N3 media containing DMEM/F12, N2 and B27 supplements, 20ug/ml Insulin, Pen/Strep (all from Invitrogen) with dox after 2 days. Cells were harvested at 48 hours, 5d and 22d post-dox induction. Control MEFs were not infected and harvested 48hr after addition of dox. For 5d and 22d, cells were FAC (Fluorescence-activated cell)-sorted for TauEGFP+ cells to select for cells that were reprogramming. To ensure that reprogramming efficiencies are comparable, immunofluorescence staining for Tuj1 was performed for each batch of cells at day 14, and only samples that average at least 20 Tuj1+ neurons per 10X field of view were used. In addition for FAC-sorted samples, only samples containing >5% TauEGFP+ cells was used.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
A detailed protocol for MethylC-seq libraries preparation can be found in Urich et al., (2015). MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing. Nat Protoc 10, 475-483.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
MethylC-seq reads were mapped to mm9 reference genome using MethylPy (https://bitbucket.org/schultzmattd/methylpy/wiki/Home). To ensure the correct calling of mCG and mCH, we removed cytosine positions with potential single nucleotide variants located at cytosines or immediately downstream of cytosines.
Genome_build: mm9
Supplementary_files_format_and_content: tab delimited text files of methylcytosine calls; columns in allc files are: column 1 - chromosome; column 2 - position; column 3 - strand; column 4 - class; column 5 - mC reads; column 6 - total reads; column 7 - methylated (Boolean value indicating the result of statistical test for methylated cytosines)
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| Submission date |
Feb 28, 2018 |
| Last update date |
Jan 15, 2019 |
| Contact name |
Joseph R Ecker |
| E-mail(s) |
ecker@salk.edu
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| Phone |
8584534100
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| Organization name |
HHMI-Salk-Institute
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| Department |
Genomic Analysis Laboratory
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| Lab |
Ecker lab
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| Street address |
10010 North Torrey Pines Road
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE111283 |
Global DNA methylation remodeling during direct reprogramming from fibroblast to neuron [MethylC-seq] |
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| Relations |
| BioSample |
SAMN08624398 |
| SRA |
SRX3752576 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3027838_allc_Ascl1_22d_B.txt.tar.gz |
3.4 Gb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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