|
| Status |
Public on Jan 28, 2020 |
| Title |
MEFdT |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Thymidine-induced MEFs by CD45 sorting
|
| Organism |
Mus musculus |
| Characteristics |
cell type: MEFs
agent: thymidine
anti-body: anti-5mC
strain: C57BL/6
|
| Treatment protocol |
4neg MEFs were seeded on mitomycin (MMC)-treated MEFs as feeder cells and cultured in DMEM with 15% FBS, 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, 0.1 mM non-essential amino acids and 100 μM thymidine as induction medium for 2 days
|
| Growth protocol |
Mouse embryonic fibroblasts (MEFs) were obtained at E14.5 embryos and supplied with Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) with 15% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin. Potential hematopoietic progenitor cells (CD45+CD41+CD31+c-Kit+) were negatively selected with Biotin labeled anti-CD45, anti-CD31, anti-CD41 and anti-c-Kit antibodies by magnetic beads.Hereafter, treated MEFs are referred to as 4neg MEFs for initiation of induction.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA samples were sheared into 200-1000 bp fragments by Bioruptor sonicator (Diagenode). Sonicated genomic DNA was immunoprecipitated by anti-5-methylcytosine antibody (Diagenode) followed by purification using Qiagen MinElute columns.
|
| Label |
Cy5
|
| Label protocol |
The purified DNA was quantified using a nanodrop ND-1000, labeled by the NimbleGen Dual-Color DNA labeling Kit and array hybridized according to the manufacturer’s protocol (Nimblegene Systems, Inc., Madison, WI, USA)
|
| |
|
| Channel 2 |
| Source name |
mouse embryonic fibroblasts
|
| Organism |
Mus musculus |
| Characteristics |
cell type: MEFs
agent: thymidine
strain: C57BL/6
chip antibody: none, input
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA samples were sheared into 200-1000 bp fragments by Bioruptor sonicator (Diagenode). Sonicated genomic DNA was immunoprecipitated by anti-5-methylcytosine antibody (Diagenode) followed by purification using Qiagen MinElute columns.
|
| Label |
Cy3
|
| Label protocol |
The purified DNA was quantified using a nanodrop ND-1000, labeled by the NimbleGen Dual-Color DNA labeling Kit and array hybridized according to the manufacturer’s protocol (Nimblegene Systems, Inc., Madison, WI, USA)
|
| |
|
| |
| Hybridization protocol |
The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
|
| Scan protocol |
Arrays were scanned on an Agilent Scanner G2505C scanner according to manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| Description |
MEFdT_MeDIP_ChIP array
|
| Data processing |
Arrays were processed using Nimblegen's standard protocol for NimbleScan v2.5 ChIP data extraction.
scaled, log2 (ChIP/Input) ratio
|
| |
|
| Submission date |
Jan 27, 2020 |
| Last update date |
Feb 03, 2020 |
| Contact name |
Benyu Liu |
| E-mail(s) |
benyuliu2014@163.com
|
| Organization name |
Institute of Biophysics, CAS
|
| Department |
The CAS key Laboratory of Infection and Immunity, Institute of Biophysics
|
| Lab |
Zusen Fan
|
| Street address |
15, Datun Road, Chaoyang district
|
| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100101 |
| Country |
China |
| |
|
| Platform ID |
GPL21852 |
| Series (1) |
| GSE144272 |
DNA methylation analysis of mouse embryonic fibroblasts (MEFs) and Thymidine treatment-induced MEFs (induced cells). |
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