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| Status |
Public on Feb 16, 2022 |
| Title |
Whole genome bisulfite sequencing of WT and Dnmt3a KO HSCs following chronic infection |
| Organism |
Mus musculus |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
Since DNMT3A is a de novo DNA methyltransferase, transcriptional differences in WT versus Dnmt3a-/- HSCs upon infection suggest a possible role for epigenetic regulation in the HSC transcriptional response to inflammation. Therefore, we wanted to investigate if differences in stress and presence of Dnmt3a affect the methylome overall, and specific IFNg responses genes.
100000 LT-HSCs (LK CD150+ CD48- were sorted into lysis buffer from the pools of naive or 1-month (M. avium) infected WT/ Dnmt3-/-_x0001_ mice (n = 7-8 per group). Around 300 ng of DNA per group was extracted with AllPrep DNA/RNA Mini Kit. NEBNext Ultra II DNA library prep kit was used for library preparation. Bisulfite conversion step was added after ‘‘methylated’’ adaptor ligation and USER excision. Then, methylated adaptor ligated DNA fragment was treated with bisulfite conversion using EZ DNA methylationlightning kit. These modified DNA fragments were then amplified with index primers with Kapa HiFi urasil+ specialized polymerase for bisulfite converted DNA. For WGBS library sequencing, 150-high output kit from illumine was used. Samples (400 million reads/ sample (15-20X coverage)) were run in NovaSeq S1 FC (2, lanes 1,600 Million reads).
Global methylation of Dnmt3a-/- cells was more divergent upon infection compared to WT. There were vastly more hyper- and hypomethylated regions in infected versus naive Dnmt3a-/- HSCs compared to WT. Among the altered regions, both WT and Dnmt3a-/- HSCs showed slightly more hypomethylated than hypermethylated regions, although changes in both directions were present. In the WT background, regions of hyper- and hypomethylation were mostly restricted to CpG islands. By contrast, highly significant differences in hypomethylation were found in the Dnmt3a-/- HSCs in CpG islands, shores, enhancers, and promoters
DNMT3A is highly active in HSCs during infection and that the loss of DNMT3A function results in significant global changes in methylation, including both hyper- and hypomethylation, in genome regions that likely affect gene transcription. we examined the methylation of Batf2, Jun, and Fos. All 3 of these prodifferentiation genes showed increased methylation in the promoter region in the Dnmt3a-/- background as compared to the WT background. Hypermethylation of these promoter regions is consistent with their transcriptional repression.
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| Overall design |
Comparison of the methylome between HSCs that are either WT or Dnmt3a KO exposed to 1 month M. avium infection
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| Contributor(s) |
Jaksik R, Kain B, Hormaechea-Agulla D, Le DT, King KY |
| Citation(s) |
33743191 |
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| Submission date |
Feb 16, 2022 |
| Last update date |
May 18, 2022 |
| Contact name |
Katherine Yudeh King |
| E-mail(s) |
kyk@bcm.edu
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| Organization name |
Baylor College of Medicine
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| Department |
Pediatrics and Infectious diseases
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| Street address |
1102 Bates Ave., Ste.1150
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| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA807680 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE196896_BCM_BS-seq1_DMR-GeneProm_v5.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
| GSE196896_RAW.tar |
811.2 Mb |
(http)(custom) |
TAR (of CGMAP) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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