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| Status |
Public on Jan 31, 2024 |
| Title |
EM-seq: Persisting epimutations iPSC con rep 1 |
| Sample type |
SRA |
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| Source name |
iPSC
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| Organism |
Mus musculus |
| Characteristics |
cell type: iPSC
treatment: Control
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| Treatment protocol |
For all cell types except PGCLCs, 1 μM BPS was added to media gassed with 5% CO2, 5% O2, and balanced N2 and then injected into sealed T-25 cell culture flasks and left to incubate for 24 hours. The media was gassed to ensure enrichment to 5% CO2, which is normally regulated by the cell incubator, limiting the potential for the pH to change in the media during this exposure period due to the absorption of CO2 by incubating cells. Following 24 hours of exposure, media containing BPS was removed and cells were washed and allowed to recover in fresh untreated media (without BPS or EtOH) for 24 hours prior to harvesting. PGCLCs were not suitable for this exposure paradigm as EpiLCs undergo differentiation to PGCLCs in cell aggregates formed in low-adherence round-bottom 96 well plates and cannot be maintained in T-25 sealed flasks. Therefore, diluted BPS was added to PGCLC media and added to cells in round-bottom 96 well plates to incubate for 24 hours prior to a shortened wash and “chase” period of 8 hours prior to cell sorting.
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| Growth protocol |
See the publication for a full description of specific growth protocols for each cell types included in the study.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from cells and smaller fragmented DNA (≤ 10 kb) and contaminating RNA were removed by cleaning the genomic DNA on a genomic DNA clean and concentratorTM-10 column (D4011), according to the manufacturer’s instructions (Zymo Research, CA USA). DNA concentration was determined using a Qubit (Q32850) and genomic DNA (100 ng) was sheered using a Bioruptor® and the size of the sheered DNA was determined using a TapeStation 4200 according to the manufacturer’s instructions in the UTSA Genomics Core (Diagenode Inc, NJ USA & Agilent Technologies, Inc CA USA).
Large size (470-520 bp) EM-seq libraries were prepared with the NEBNext® Enzymatic Methyl-seq kit (E7120S) according to the manufacturer’s protocol (New England Biolabs, MA USA). Briefly, this process consisted of A-tailing, adaptor ligation, DNA oxidation by TET2 initiated by the addition of Fe (II), strand denaturization with formamide, deamination by APOBEC3A, polymerase chain reaction amplification, and bead selection to generate the final libraries. Distinct index adaptors were used for multiplexing samples across multiple sequencing lanes.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
EM-seq data was processed using the comprehensive wg-blimp v10.0.0 software pipeline. In brief, reads were trimmed prior to initiating the wg-blimp pipeline using Trim Galore. Sequenced reads were aligned to the mm10 genome using gemBS. The BAM files from alignment underwent a series of QC tests including read quality scoring by FastQC, overall and per-chromosome read coverage calculation, GC content, duplication rate, clipping profiles by Qualimap, and deduplication by the Picard toolkit. Methylation calling was performed by MethylDackel and statistically significant DMC/DMR calling was performed by the metilene and BSmooth algorithms. Metilene uses a binary segmentation algorithm combined with a two-dimensional statistical test that allows the detection of DMCs/DMRs in large methylation experiments with multiple groups of samples. Finally, potential regulatory regions were identified through the use of MethylSeekR. Results from the pipeline were displayed in the wg-blimp interactive results web browser that was built using the R Shiny local browser hosting framework.
Assembly: mm10
Supplementary files format and content: bedGraph
Library strategy: EM-seq
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| Submission date |
Jan 04, 2024 |
| Last update date |
Jan 31, 2024 |
| Contact name |
Jake Dean Lehle |
| E-mail(s) |
jakelehle@gmail.com
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| Phone |
5129928144
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| Organization name |
The University of Texas at San Antonio
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| Department |
NDRB
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| Lab |
McCarrey
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| Street address |
16631 Vance Jackson Rd APT#1220
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| City |
San Antonio |
| State/province |
TX |
| ZIP/Postal code |
78249 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE252520 |
Endocrine disruptor-induced epimutagenesis in vitro: Insight into molecular mechanisms [EM-Seq] |
| GSE252723 |
Endocrine disruptor-induced epimutagenesis in vitro: Insight into molecular mechanisms |
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| Relations |
| BioSample |
SAMN38050661 |
| SRA |
SRX22318441 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8002497_iPSC_E_1_S1_CpG.bedGraph.gz |
163.8 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
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