|
| Status |
Public on Jul 02, 2020 |
| Title |
3730CHDI_820487_RRBS |
| Sample type |
SRA |
| |
|
| Source name |
Cerebellum
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Q175
tissue: Cerebellum
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA methylation at CpG sites was assayed with a modified version of Reduced Representation Bisulfite Sequencing (RRBS; Meissner et al., 2008). RRBS was carried out by the HudsonAlpha Institute for Biotechnology. Genomic DNA is isolated from each cell line using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations for each genomic DNA preparation are determined using fluorescent DNA binding dye and a fluorometer (Invitrogen Quant-iT dsDNA High Sensitivity Kit and Qubit Fluorometer). 1 µg of DNA was used to make an RRBS library.
RRBS library construction starts with MspI digestion of genomic DNA , which cuts at every CCGG regardless of methylation status. Klenow exo- DNA Polymerase is then used to fill in the recessed end of the genomic DNA and add an adenosine as a 3prime overhang. Next, a methylated version of the Illumina paired-end adapters is ligated onto the DNA. Adapter ligated genomic DNA fragments between 105 and 185 basepairs are selected using agarose gel electrophoresis and Qiagen Qiaquick Gel Extraction Kit. The selected adapter-ligated fragments are treated with sodium bisulfite using the Zymo Research EZ DNA Methylation Gold Kit, which converts unmethylated cytosines to uracils and leaves methylated cytosines unchanged. Bisulfite treated DNA is amplified in a final PCR reaction which has been optimized to uniformly amplify diverse fragment sizes and sequence contexts in the same reaction. During this final PCR reaction uracils are copied as thymines resulting in a thymine in the PCR products wherever an unmethylated cytosine existed in the genomic DNA. The sample is now ready for sequencing on the Illumina sequencing platform. These libraries were sequenced with an Illumina Genome Analyzer IIx according to the manufacturer's recommendations.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
The provided FASTQ files are not the raw FASTQ files from the sequencer; they have been processed by adapter trimming, and further processed using a custom script to make the file suitable for alignment.
|
| Data processing |
To analyze the sequence data, a reference genome is created that contains only the 36 base pairs adjacent to every MspI site and every C in those sequences is changed to T. A converted sequence read file is then created by changing each C in the original sequence reads to a T. The converted sequence reads are aligned to the converted reference genome, and only reads that map uniquely to the reference genome are kept. Once reads are aligned the percent methylation is calculated for each CpG using the original sequence reads. The percent methylation and number of reads is reported for each CpG.
Genome_build: mm10
|
| |
|
| Submission date |
Mar 18, 2020 |
| Last update date |
Jul 04, 2020 |
| Contact name |
Steve Horvath |
| E-mail(s) |
shorvath@altoslabs.com
|
| Organization name |
University of California, Los Angeles
|
| Department |
Human Genetics
|
| Lab |
Horvath
|
| Street address |
695 Charles E. Young Drive South, Box 708822
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095-7088 |
| Country |
USA |
| |
|
| Platform ID |
GPL11002 |
| Series (2) |
| GSE147004 |
DNA Methylation Study of Huntington's Disease and Motor Progression in Three Species |
| GSE147156 |
Reduced Representation Bisulfite Sequencing from HD knock-in mouse model |
|
| Relations |
| BioSample |
SAMN14396832 |
| SRA |
SRX7945577 |
