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| Status |
Public on Nov 01, 2016 |
| Title |
Uhrf1_Heterozygous_3 |
| Sample type |
SRA |
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| Source name |
E16 full cortex
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: Uhrf1 +/-
age: E16
tissue: full cortex
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| Growth protocol |
dissection of the entire cortex in E16 mice
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Samples were prepared according to the protocol from the NextFlex kit from Bioo Scientific. An oxidation step was added to remoce 5-hydroxymethylcytosine. Briefly 1 µg of mouse DNA was digested with MSPI (NEB Biolabs) in Buffer 4 at 37°C O/N. Then, the digested DNA was cleaned up with the MinElute® Reaction Cleanup Kit (Qiagen) according to the manufacturer´s instructions. Briefly, 300 µl buffer ERC were added to the MSPI digest, applied to a spin column provided in the kit and centrifugation was performed for 1 minute at 17,900 x g. Then, the column was washed with 750 µl buffer PE, dried by centrifugation for 1 minute at 17,900 x g and the DNA was eluted with 18 µl buffer EB. DNA concentration was determined with a NanoDrop 2000 spectrophotometer (peqlab). 500 ng of digested and purified DNA were used for the library preparation procedure with the NEXTflex™ Bisulfite Library Prep Kit (BIOO Scientific). Library preparation was performed according to the manufacturer´s instructions with some modifications. Specifically, to avoid any false positives through changes in hydroxymethylation, a DNA oxidation step was included. Briefly, end repair was performed with 500 ng digested, purified DNA in end repair buffer mix and end repair enzyme mix in a total volume of 50 µl. The reaction was incubated at 22°C for 30 minutes and then cleaned up with the MinElute® Reaction Cleanup Kit as described above. Then, 16.5 µl of the eluate were mixed with 4.5 µl of adenylation mix and the reaction was incubated for 30 minutes at 37 °C. Afterwards, 31.5 µl ligation mix and 2.5 µl of individual adapters (diluted 1:2) were added, and adapter ligation was performed for 15´ at 22°C. All control PCR products were ligated to a separate adapter, to avoid any mix up between samples and controls. Then, a gel free size selection was performed. 44 µl of Agencourt AMPure XP Beads (Beckman Coulter) were added to the reaction and DNA was allowed to bind to the beads for 5 minutes at RT. Then, the reaction tubes were placed on a magnetic stand and the beads were allowed to be separated from the solution for 5 minutes. Afterwards, the supernatant was discarded and two washes with 200 µl 80 % Ethanol were performed. Then, the beads were allowed to dry for 3 minutes and resuspended in 52 µl resuspension buffer. The beads were incubated for 2 minutes at RT. Then, the reaction tubes were placed on a magnetic stand and the beads were allowed to be separated from the solution for 5 minutes. Then, 50 µl of the supernatant containing the DNA was transferred into a new well, 25 µl Agencourt AMPure XP Beads were added to the reaction and incubated for 5 minutes at RT. Then, the reaction tubes were placed on a magnetic stand and the beads were allowed to be separated from the solution for 5 minutes. Afterwards, 73 µl of the supernatant containing the DNA of interest was transferred into a new well and again 14 µl Agencourt AMPure XP Beads were added to the reaction and incubated for 5 minutes at RT. Then, the reaction tubes were placed on a magnetic stand and the beads were allowed to be separated from the solution for 5 minutes. The supernatant was discarded and two washes with 200 µl 80% Ethanol were performed. Afterwards, the beads were dried for 15 minutes at RT, to ensure that all ethanol had been removed. Then, the beads were resuspended in 25 µl sterile water (Carl Roth) and incubated for 15 minutes at RT. Then, the reaction tubes were placed on a magnetic stand and the beads were allowed to be separated from the solution for 5 minutes. Afterwards the DNA was prepurified with Micro-Bio-Spin P-6 SCC columns (Bio-Rad) as has been described in [3]. Briefly, 24 µl sample were transferred to a spin column that had been washed with 500 µl sterile water four times. The DNA was purified by centrifugation through the spin column for 8 minutes at 1000 x g at RT. Oxidation was performed as with KRuO4 (Sigma-Aldrich) [4] as has been described by others [3]. First, 21.75 µl DNA were mixed with 1.25 µl extra pure NaOH (Carl Roth) and incubated at 37°C and 300 rpm on a shaking incubator. Then, the DNA was snap cooled on ice for 5 minutes and 20 µl KRuO4 solution (15mM in 0.05 M NaOH) was added. The DNA was oxidized for 1 h on ice, and purified again with Micro-Bio-Spin P-6 SCC columns as described above. Then, bisulfite conversion of the DNA was performed with the EZ Methylation Gold Kit (Zymo Research) according to the manufacturer´s instructions. Briefly, 130 µl conversion reagent were added to 20 µl oxidized and purified DNA. The reaction was incubated for 10 minutes at 98°C and for 2.5 hours at 64°C. Then, the samples were loaded on spin columns containing 600 µl M-Binding buffer and mixed by inverting. The DNA was bound to the column by centrifugation for 30 seconds at 18620 x g. Then, the column was wahed with 100 µl wash buffer, and 200 µl desulphonation buffer were added. The desulphonation buffer was incubated for 17 minutes at RT, and then removed by centrifugation. The column was washed twice with 200 µl wash buffer, and dried by centrifugation for 10 seconds. Finally, 17 µl elution buffer were added to the column, incubated for 1 minute and the DNA was eluted by centrifugation. Afterwards, PCR amplification of the oxidized and bisulfite converted libraries was performed with PfuCx Hot Start (Agilent). 15 µl DNA were amplified with the NEXTflex™ Primer Mix. Cycling conditions were: Initial denaturation for five minutes at 95°C. Then 18 cycles of 95°C for 30 seconds, 65°C for 30 seconds and 72°C for 45 seconds. Afterwards a final extension at 72°C for 7 minutes. The libraries were purified again using AMPure XP beads (Beckmann Coulter). Finally, quality control with a Bioanalyzer® (Agilent) and sequencing with a HiSeq 1500 sequencer (Illumina Inc.)
RRBS including an oxidation step to remove 5-hydroxymethylcytosine
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Image analysis and base calling resulted in .bcl files, which were converted into .fastq files by the CASAVA1.8.2 software
Fastq files were imported to the commercially available Genomatix mining station with the data import and validation tool ver. 2.2.2
Reads were mapped to the mouse genome version GRCm38 with the commercially available Genomatix mapper ver. 3.7.6.3 allowing no mismatch
Methylation per CpG was calculated with the commercially available Genomatix mining station
CpGs were filtered for a minimal coverage > 5 with the free software R ver 3.2.1
Merging of the individual files and further analyzes were performed with the R package methylkit
Genome_build: GRCm38
Supplementary_files_format_and_content: tab delimited .txt file with methylation per CpG, columns are column1: chr.base column2: chromosome column3: base column 4: strand column5: coverage column6: frequency C column7: frequency T
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| Submission date |
Jul 19, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gunnar Schotta |
| E-mail(s) |
gunnar.schotta@bmc.med.lmu.de
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| Phone |
089218075422
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| Organization name |
LMU Munich
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| Department |
Biomedical Center
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| Street address |
Grosshaderner Strasse 9
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| City |
Planegg-Martinsried |
| ZIP/Postal code |
82152 |
| Country |
Germany |
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| Platform ID |
GPL18480 |
| Series (2) |
| GSE84549 |
Loss of Uhrf1 in neural stem cells leads to activation of retroviral elements and delayed neurodegeneration [RRBS] |
| GSE84550 |
Loss of Uhrf1 in neural stem cells leads to activation of retroviral elements and delayed neurodegeneration |
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| Relations |
| BioSample |
SAMN05415228 |
| SRA |
SRX1959679 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2241743_Uhrf1_Heterozygous_3.txt.gz |
9.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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