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| Status |
Public on Jan 04, 2021 |
| Title |
The hepatic methylome of liver-specific insulin receptor knockout (LIRKO) mouse offspring |
| Organism |
Mus musculus |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
Liver-specific insulin receptor knockout (LIRKO) mouse is a unique non-dietary model of insulin resistant, hyperglycemia, dyslipidemia and atherosclerosis that resembles several clinical features of the human metabolic syndrome. By enhanced reduced representation bisulfite sequencing (ERRBS) analysis of the livers of wild-type (WT) offspring of LIRKO mice, we identified that genes with differential DNA methylation were enriched for cholesterol synthesis, MAPK, AKT, insulin and TGF-β signaling, including the NREP and GDF15.
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| Overall design |
LIRKO (insulin receptor-IRlox/lox; Albumin-Cre+/-) mice were generated as previously described. The control offspring group consisted of the F1 offspring from a control male and female (insulin receptor-IRlox/lox; Albumin-Cre-/-). Control parents were crossed for 4 generations for minimizing any epigenetic memory from the presence of Cre. Father LIRKO offspring (FL) resulted from the crossing of a male LIRKO with a control female. Mother LIRKO offspring (ML) resulted from the breeding of a control male with a LIRKO female. High molecular weight DNA was isolated from control, FL and ML offspring livers using the Gentra puregene tissue kit (Qiagen). ERRBS library preparation, sequencing, and post-processing of the raw data were performed at the Epigenomics Core at Weill Cornell Medicine. Briefly, 75 ng of DNA (>20kb in size) were digested with MspI. After phenol extraction and ethanol precipitation MspI ends were end-repaired, A-tailed and ligated to methylated TruSeq adapters (Illumina Inc. San Diego, CA). Samples were size selected in a 1.5% agarose gel and two size fragment lengths of 150–250 bp and 250–400 bp were gel isolated. These two fractions were subjected to overnight bisulfite conversion (55 cycles of 95 °C for 30 sec, 50 °C for 15 min) using EZ DNA methylation kit (Zymo Research, Irvine CA). Purified bisulfite converted DNA was PCR amplified using TruSeq primers (Illumina Inc. San Diego, CA) for 18 cycles of denaturing, annealing and extension/elongation steps: 94 °C for 20 secs, 65 °C for 30 secs, 72 °C for 1 min, followed by 72 °C for 3 min. The resulting libraries were normalized to 2 nM and pooled at the same molar ratio. The final samples were pooled according to the desired plexity, clustered at 6.5 pM on the single-read flow cell and sequenced for 50 cycles on an Illumina HiSeq 2500. The primary processing of sequencing images was done using Illumina’s Real Time Analysis software (RTA). Illumina’s BCL2FASTQ version 2.17 software was then used to de-multiplex samples and generate raw reads and respective quality scores. For analysis of bisulfite-treated sequence reads (with an average bisulfite conversion rate of >99% for all samples), reads were filtered for adapter sequences using the FLEXBAR software. The adapter sequence present in the 3’ end of the reads was removed if it aligned with the adapter sequence at least 6 bps and had at most a 0.2 mismatch error rate. Reads were aligned to the whole genome using the Bismark alignment software with a maximum of two mismatches in a directional manner and only uniquely aligning reads were retained. In order to call a methylation score for a base position, read bases aligning to that position had at least a 20 phred quality score and that base position had at least 10x coverage. The percentage of bisulfite converted cytosines (representing unmethylated cytosines) and non-converted cytosines (representing methylated cytosines) were recorded for each cytosine position in CpG, CHG, and CHH contexts (with H corresponding to A, C, or T nucleotides).
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| Contributor(s) |
De Jesus DF, Kulkarni RN |
| Citation(s) |
32250344 |
| |
| Submission date |
Dec 03, 2019 |
| Last update date |
Jan 04, 2021 |
| Contact name |
Hui Pan |
| E-mail(s) |
Hui.Pan@joslin.harvard.edu
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| Organization name |
Joslin Diabetes Center
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| Department |
Bioinformatics and Biostatistics Core
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| Street address |
1 Joslin Place
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| City |
Boston |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platforms (1) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA593236 |
| SRA |
SRP234561 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE141379_chg.189_DNA_Liver.mincov10.txt.gz |
18.4 Mb |
(ftp)(http) |
TXT |
| GSE141379_chg.383_DNA_Liver.mincov10.txt.gz |
15.6 Mb |
(ftp)(http) |
TXT |
| GSE141379_chg.C30_DNA_Liver.mincov10.txt.gz |
16.8 Mb |
(ftp)(http) |
TXT |
| GSE141379_chg.C81_DNA_Liver.mincov10.txt.gz |
18.0 Mb |
(ftp)(http) |
TXT |
| GSE141379_chg.FL12_DNA_Liver.mincov10.txt.gz |
20.2 Mb |
(ftp)(http) |
TXT |
| GSE141379_chg.FL15_DNA_Liver.mincov10.txt.gz |
18.5 Mb |
(ftp)(http) |
TXT |
| GSE141379_chg.FL2_DNA_Liver.mincov10.txt.gz |
14.0 Mb |
(ftp)(http) |
TXT |
| GSE141379_chg.FL6_DNA_Liver.mincov10.txt.gz |
17.4 Mb |
(ftp)(http) |
TXT |
| GSE141379_chg.ML13_DNA_Liver.mincov10.txt.gz |
16.9 Mb |
(ftp)(http) |
TXT |
| GSE141379_chg.ML1_DNA_Liver.mincov10.txt.gz |
16.8 Mb |
(ftp)(http) |
TXT |
| GSE141379_chg.ML21_DNA_Liver.mincov10.txt.gz |
17.1 Mb |
(ftp)(http) |
TXT |
| GSE141379_chg.ML6_DNA_Liver.mincov10.txt.gz |
16.9 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.189_DNA_Liver.mincov10.txt.gz |
41.9 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.383_DNA_Liver.mincov10.txt.gz |
34.9 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.C30_DNA_Liver.mincov10.txt.gz |
38.5 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.C81_DNA_Liver.mincov10.txt.gz |
41.3 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.FL12_DNA_Liver.mincov10.txt.gz |
46.9 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.FL15_DNA_Liver.mincov10.txt.gz |
42.0 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.FL2_DNA_Liver.mincov10.txt.gz |
31.7 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.FL6_DNA_Liver.mincov10.txt.gz |
39.2 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.ML13_DNA_Liver.mincov10.txt.gz |
38.4 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.ML1_DNA_Liver.mincov10.txt.gz |
37.9 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.ML21_DNA_Liver.mincov10.txt.gz |
38.8 Mb |
(ftp)(http) |
TXT |
| GSE141379_chh.ML6_DNA_Liver.mincov10.txt.gz |
38.1 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.189_DNA_Liver.mincov10.txt.gz |
16.1 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.383_DNA_Liver.mincov10.txt.gz |
16.0 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.C30_DNA_Liver.mincov10.txt.gz |
14.9 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.C81_DNA_Liver.mincov10.txt.gz |
16.1 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.FL12_DNA_Liver.mincov10.txt.gz |
16.0 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.FL15_DNA_Liver.mincov10.txt.gz |
16.5 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.FL2_DNA_Liver.mincov10.txt.gz |
11.8 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.FL6_DNA_Liver.mincov10.txt.gz |
16.4 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.ML13_DNA_Liver.mincov10.txt.gz |
16.6 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.ML1_DNA_Liver.mincov10.txt.gz |
16.7 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.ML21_DNA_Liver.mincov10.txt.gz |
16.6 Mb |
(ftp)(http) |
TXT |
| GSE141379_cpg.ML6_DNA_Liver.mincov10.txt.gz |
16.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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