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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 27, 2023 |
| Title |
mouse kidney, UUO7D, 5AZA, sample 1 [5aza1] |
| Sample type |
SRA |
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| Source name |
Kidney
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| Organism |
Mus musculus |
| Characteristics |
tissue: Kidney
strain: C57BL/6J
Sex: male
genotype: wild type
treatment: UUO+5AZA
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| Treatment protocol |
For UUO surgery, mice were anesthetized and the left ureter was ligated twice with suture and a cut was made between the two ligations. The sham-operated mice experienced similar surgery operations but without ureter ligation . For 5AZA treatment, 1mg/kg/day 5AZA in saline was administered through I.P. injection.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The genomic DNA samples were extracted from mouse kidney cortex and outer medulla with QIAmp DNA Blood Mini kit from Qiagen (Germantown, MD) according to the manufacturer’s manual.
RRBS was performed according to a previously published protocol (Gu et al. 2010; Meissner et al. 2008) with minor modifications. For each sample, 1 g genomic DNA was first digested overnight using 20 units of MspI (New England Biolabs, NEB). The digested DNA was end-repaired and adenylated in a 20 l reaction consisting of 10U of Klenow fragments (exo-, Enzymatics, MA, USA), and 2 l premixed nucleotide triphosphates (1 mM dGTP, 10 mM dATP, 1 mM methylated dCTP). The reaction was incubated at 30 C for 30 min followed by 37 C for an additional 30 min. The methylated Illumina adapters were ligated with adenylated DNA fragments in a 20 l reaction containing 1 l concentrated T4 ligase (Enzymatics) at room temperature for 15 minutes. The ligation products were gel-selected for fragments with insertion sizes of 40 to 120 bp and 120 to 220 base pairs as previously suggested (Gu et al. 2010; Meissner et al. 2008). Bisulfite treatment of the sequencing library was conducted using the EZ DNA methylation kit (Zymo Research, Irvine, CA) according to manufacturer's protocol. The final libraries were generated by PCR amplification of 5 ?m of bisulfite-converted template using PfuTurbo Cx Hotstart polymerase and Illumina pair-end PCR primers (PE1.0 and PE2.0).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
To map the sequencing reads from RRBS, the mouse genome (mm9) was indexed by converting all C’s and G’s to T’s and A’s, respectively, which results in two different reference databases representing both strands. The cleaned reads was used to map to each of the two reference databases using Bowtie (Langmead et al., 2009) after converting all C’s to T’s. For each read, an in-house script computed the best of all alignments for the different loci using two different reference databases based on the number of mismatches after realigning the original read and reference sequences. Another in-house script determined the methylation status of each cytosine residue by comparing the bisulfite-converted sequence to the reference sequence. The third script piled reads for each cytosine in the reference genome and counted the numbers of reads that contained methylated and unmethylated cytosines, respectively. Finally the methylation of each CpG site was defined as the fraction of methylated reads to that of methylated and unmethylated reads combined. CpGs with < 5 reads were filtered out for further analyses. We used windows of length 200bp with an overlap of 100bp between to identify differentially methylated regions (DMRs) by summing the numbers of methylated and unmethylated CpGs in reads, respectively.
Methylation calculation: The methylation level [(# of methylated)/(# of methylated + # of unmethylated)] was calculated after measuring the amount of methylation (CG->CG) and unmethylation (CG->TG) in the CpG position. Only CpGs that are covered by at least 10 sequencing reads were included.
Assembly: mm9
Supplementary files format and content: bed
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| Submission date |
Oct 27, 2023 |
| Last update date |
Nov 27, 2023 |
| Contact name |
Qingqing Wei |
| E-mail(s) |
QWEI@AUGUSTA.EDU
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| Phone |
7067213551
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| Organization name |
Augusta University
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| Department |
Cellular Biology & Anatomy
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| Lab |
CB1124
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| Street address |
1460 Laney Walker Blvd.
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| City |
AUGUSTA |
| State/province |
Georgia |
| ZIP/Postal code |
30912 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE246417 |
Hypermethylation leads to the loss of HOXA5, resulting in JAG1 expression and NOTCH signaling for renal fibrosis |
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| Relations |
| BioSample |
SAMN38022073 |
| SRA |
SRX22253266 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7868591_5aza1.best.unique.cg.bed.gz |
12.2 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
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