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| Status |
Public on Jan 01, 2018 |
| Title |
WT_eRRBS, biological replicate 2 |
| Sample type |
SRA |
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| Source name |
DNMT TKO ES Cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: ES Cells
genotype: Dnmt1, Dnmt3a and Dnmt3b triple knockout stably expressing DNMT3A WT
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| Treatment protocol |
TKO ES cells were transfected by Lipofectamine 2000 (Invitrogen) with the pPyCAGIZ empty vector (EV) or that carrying wild-type (WT) or mutant DNMT3A. At 48 hours following transfection, the transduced ES cells were selected out in 50 µg/ml Zeocin (Invitrogen) for 10 days and continuously maintained in the medium with 25 µg/ml Zeocin.
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| Growth protocol |
Dnmt3a, Dnmt3b and Dnmt1 triple knockout (TKO) mouse ES cells were cultivated on gelatin-coated dishes in the high-glucose DMEM base medium (Invitrogen) supplemented with 15% of fetal bovine serum (FBS, Invitrogen), 1 x nonessential amino acids (Invitrogen), 0.1 mM 2-mercaptoethanol, and 1000 U/ml leukemia inhibitory factor (ESGRO).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen).
eRRBS was carried out with a previously described protocol with slight modification (Garrett-Bakelman et al., 2015). Briefly, ~300 ng of genomic DNA were digested with three different enzymes (~80 units of MspI, 40 units of BfaI and 40 units of MseI) to enhance genomic fragmentation and coverage. The produced fragments were ligated to pre-annealed adapters containing 5’-methyl-cytosine instead of cytosine, followed by overhang fill-in, 3’-terminal-A extension and purification. Bisulfite treatment of the fragments was done using the EZ DNA Methylation–Lightning kit (Zymo Research). Amplified eRRBS libraries was quality checked with Agilent 2200 TapeStation, followed by deep sequencing on the Illumina HiSeq-2000 genome analyzer with 50 bp PE parameters.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
TKO ES cells re-expressing WT DNMT3A
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| Data processing |
Sequence reads from bisulfite-treated libraries were identified using standard Illumina base-calling software.
Residual cytosines (Cs) in each read were first converted to thymines (Ts), with each such conversion noted for subsequent analysis. A reference sequence database was constructed from the 50-bp ends of each computationally predicted MspI-TaqI fragment in the 40–350 bp size range.
All Cs in each fragment end were then converted to Ts; the converted reads were aligned to the converted reference by Bismark with Bowtie in the non-directional mode. The number of mismatches in the induced alignment was then counted between the unconverted read and reference, ignoring cases in which a T in the unconverted read is matched to a C in the unconverted reference. For a given read, the best alignment was kept. If there was more than one best alignment, the read was discarded as non-unique.
The methylation level of each sampled cytosine was estimated as the number of reads reporting a C, divided by the total number of reads reporting a C or T.
Genome_build: mm9
Supplementary_files_format_and_content: bed files contain all mapped Cs with chr, start, end, and methylated reads/total reads (percentage) listed.
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| Submission date |
Nov 01, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Rui Lu |
| E-mail(s) |
ruilu1@uabmc.edu
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| Organization name |
Univ of Alabama at Birmingham
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| Street address |
1824 6th Ave S
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| City |
Birmingham |
| State/province |
AL |
| ZIP/Postal code |
35294 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE99390 |
Structural basis for DNMT3A-mediated de novo DNA methylation (eRRBS) |
| GSE99391 |
Structural basis for DNMT3A-mediated de novo DNA methylation |
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| Relations |
| BioSample |
SAMN07964396 |
| SRA |
SRX3350067 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2837151_eRRBS_WT2_meth.bed.gz |
1.8 Gb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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