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| Status |
Public on Dec 19, 2017 |
| Title |
HF3, RNAseq |
| Sample type |
SRA |
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| Source name |
colonic epithelium
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| Organism |
Mus musculus |
| Characteristics |
Sex: male
age: six-week old
strain: C57BL/6
tissue: colon
diet: 60% fat diet (HF)
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| Treatment protocol |
Six-week old male C57BL/6 mice were placed on three different dietary regimens as follows: 1) low-fat diet for 20 weeks; 2) high-fat diet for 20 weeks; 3) high-fat diet for an initial 15 weeks then switched to low-fat diet for another 5 weeks.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The entire colon was removed from mouse and colonic epithelium was isolated using PBS with 5 mM EDTA. Genomic DNA and total RNA were isolated using Qiagen AllPrep DNA/RNA/miRNA Universal Kit according to the manufacturer’s instructions.
Whole-genome bisulfite sequencing libraries were prepared as follows: genomic DNA was sonicated to an average size of 200 bp using Covaris S220. DNA fragments were end-repaired, adenylated, and ligated to Illumina-compatible adaptors using BIOO NEXTflex Bisulfite-Seq Kit. Bisulfite conversion was performed using EZ DNA Methylation-Lightning™ Kit (Zymo Research Corporation) according to the manufacturer’s instructions. Then PCR was performed to enrich bisulfite converted and adaptor-ligated fragments. RNA sequencing libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, #RS-122-2103). Bisulfite PCR sequencing libraries were prepared as follows: Genomic DNA was bisulfite converted using EZ DNA Methylation-Lightning™ Kit (Zymo Research Corporation) prior to PCR amplification. Amplicons from a single sample were pooled and individually indexed using TruSeq™ RNA Sample Preparation kit (Illumina) to create a multiplex library.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Each raw dataset was filtered to remove any read pairs with average base quality score <20. Cutadapt (parameters: --adapter=AGATCGGAAGAGC -O 5) was used to trim adapters from BSP-seq libraries; adapter trimming was deemed unnecessary for the RNA-seq and WGBS libraries. A minimum fragment length of 30nt (post-trimming) was required.
RNA-seq reads were mapped against the mm10 reference genome via TopHat v2.0.4 (parameters: --b2-sensitive --library-type fr-firststrand -g 10 --mate-inner-dist 40 --mate-std-dev 50).
RNA-seq read counts per gene per sample were collected using HTSeq-Count.
Differentially expressed genes were determined using DESeq2 v1.2.10 , with threholds p-value <0.01, adjusted p-value < 0.25, and fold change > 1.2.
RNA-seq coverage tracks (per genomic strand) were generated with bedtools2 v2.19.0 genomeCoverageBed, then convert to bigWig format with UCSC command-line tool bedGraphToBigWig.
BSP-seq reads were mapped via Bismark v0.7.8 (parameters: -I 0 -X 1000 --non_bs_mm --chunkmbs 1024 --non_directional) against a masked version of the mm10 reference genome where are nucleotides in non-query regions were replaced with Ns.
BSP-seq alignments were post-processed to clip redundant mapped bases due to overlapping mates from the same read pair.
Percent methylation at each CpG site in the query regions of the BSP-seq data was calculated as methylated mapped C bases divided by total (methylated+unmethylated) mapped C bases.
BSP-seq coverage tracks were generated with bedtools2 v2.19.0 genomeCoverageBed, then convert to bigWig format with UCSC command-line tool bedGraphToBigWig.
WGBS reads were mapped against the mm10 reference genome via Bismark v0.14.0 (parameters: -I 0 -X 10000 --non_bs_mm --sam --old_flag -n 2 -l 50 -e 70 --chunkmbs 1024), then deduplicated with deduplicate_bismark v0.14.0 (parameters: --paired).
WGBS alignments were post-processed to separate hits according to source genome strand, to clip redundant mapped bases due to overlapping mates from the same read pair, and to clip 5' and/or 3' read cycles with methylation bias as identified with bismark_methylation_extractor using the --mbias_only option.
WGBS coverage tracks (per genomic strand) were generated with bedtools2 v2.19.0 genomeCoverageBed, then convert to bigWig format with UCSC command-line tool bedGraphToBigWig.
WGBS percent methylation was calculated (per cytosine in CpG, per genomic strand) as methylated mapped C bases divided by total (methylated+unmethylated) mapped C bases.
Differentially methylated regions were identified by merging CpG sites with differential methylation according to the Rao Scott Likelihood Ratio Test (p-value 0.05) within 5kb, with minimum weighted coverage of 10 in at least one cohort. DMRs were then filtered to require at least 2 CpG sites, region FDR <0.01, and methylation difference of least 30% or 5-fold.
Genome_build: mm10
Supplementary_files_format_and_content: RNAseq mapped read depth by strand in bigWig format.
Supplementary_files_format_and_content: Tab-delimited text file with read counts per gene, per sample.
Supplementary_files_format_and_content: Tab-delimited text files with DESeq2 values (fold change, p-value, adjusted p-value) and DEG status according to selected thresholds.
Supplementary_files_format_and_content: BSPseq mapped read depth in bigWig format.
Supplementary_files_format_and_content: Tab-delimited text file with percent methylation at each CpG site in the BSP-seq query regions.
Supplementary_files_format_and_content: WGBS mapped read depth by strand in bigWig format.
Supplementary_files_format_and_content: WGBS percent methylation per CpG site in bigWig format; C on plus strand have positive values, C on minus strand have negative values;
Supplementary_files_format_and_content: WGBS DMRs for high fat vs low fat diet conditions.
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| Submission date |
Aug 17, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Paul A Wade |
| E-mail(s) |
wadep2@niehs.nih.gov
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| Phone |
919-541-3392
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| Organization name |
NIEHS
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| Department |
Laboratory of Molecular Carcinogenesis
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| Street address |
111 TW Alexander Drive
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| City |
Research Triangle Park |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE85731 |
Epigenetic memory of obesity in mouse colonic epithelium |
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| Relations |
| BioSample |
SAMN05583591 |
| SRA |
SRX2033393 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2283275_HF3.RNAseq.minus_strand_mapped_depth.bigWig |
69.5 Mb |
(ftp)(http) |
BIGWIG |
| GSM2283275_HF3.RNAseq.plus_strand_mapped_depth.bigWig |
70.5 Mb |
(ftp)(http) |
BIGWIG |
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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