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| Status |
Public on Feb 22, 2014 |
| Title |
Ad_Front_NeuN_pos.TARRBS |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Homo sapiens |
| Characteristics |
tissue: frontal lobe
developmental stage: adult
cell type: NeuN+ neurons
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
500 mg of cryopreserved human left frontal cortex was homogenized on ice by douncing in 5 ml lysis buffer (0.32 M Sucrose, 5 mM CaCl2, 3 mM, 0.1mM EDTA, 10 mM Tris-HCl (pH 8.0), 1 mM DTT, and 0.1% TritonX-100). Homogenates were transferred to ultracentrifuge tube, with Sucrose Solution (1.8 M Sucrose, 3 mM Mg(Ac)2, 1 mM DTT, and 10 mM Tris-HCl (pH 8.0)) carefully pipetted at the bottom of the tube to form a concentration gradient. Ultracentrifugation was performed at 100,000 g for 2.5 hrs at 4°C. After centrifuge, supernatant including debris was removed and the pellet was incubated in 0.5 ml cold PBS on ice for 20 min before thoroughly triturated by pipetting. Immunostaining mix was prepared by combining 300 µl PBS, 1.2 µg NeuN antibody (Millipore, MAB377), 200 µl Blocking Mix ( 2.5% BSA and 10% Goat Serum in PBS), and 2 µg of Alexa Fluor 488 conjugated secondary antibody (Cell Signaling, #4488) together and rotated for 5 min at room temperature in the dark. An isotype antibody control was processed in parallel by adding the same amount of mouse IgG instead of NeuN antibody. Then 1 ml nuclei suspension was added, and the mixture was incubated by rotating in the cold room for 45 min. After incubation, samples were retrieved and stained with Hoechst 33342 for another 2 min. The immunostaining result was checked under the microscope. Immumotagged samples were diluted 10 times in PBS and filtered through a 40μm filter before loaded to the FACS machine. A preliminary run was performed to gate out the proper nuclei size and the fluorescence bright population before the sort which separate the NeuN+ nuclei. Once the sort was done, a small amount of sorted sample was run again through the instrument to confirm the purity. After FACS, PBS was added to raise the volume of the sort to 10 ml. Then the sorted sample was mixed with 2 ml Sucrose Solution, 50 µl CaCl2 (1 M), and 30 µl Mg(Ac)2 (1 M), and incubated on ice for 15 min before centrifuged at 1786 g for 15 min at 4°C. The NeuN+ nuclei pellet was resuspended in PBS. DNA was extracted by Qiagen Blood & Cell Culture DNA Kits following the manufacture’s instructions.
Genomic DNA extracted from purified neuron nucleus were first digested with MspI for 3 hours at 37 °C and purified using QIAquick columns. Then, glycosylation and oxidation of the DNA was performed similar to the TAB-Seq, except for an additional size selection of 180-600 bp fragments on a 2% agarose gel was performed after the adapter ligation.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Base calling is performed by CASAVA_v1.8.2
Alignment is performed by Bismark v0.7.6
Convert alignment results to information of each base of reference sequence, using SAMTools mpileup v0.1.18 (r982:295)
Calculate modification p-value of each site using R (version 2.14.2) function dbinom(), then assess FDR using Benjamini-Hochberg method
Genome_build: hg19
Supplementary_files_format_and_content: hmC modification level of each site
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| Submission date |
Feb 21, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Xianlong Li |
| E-mail(s) |
acelixianlong@gmail.com
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| Organization name |
Peking University
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| Department |
Biodynamic Optical Imaging Center, College of Life Sciences
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| Street address |
5, Yiheyuan Rd., Haidian District
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| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE55249 |
Whole-genome analysis of 5-hydroxymethylcytosine and 5-methylcytosine at base resolution in the human brain |
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| Relations |
| BioSample |
SAMN02649410 |
| SRA |
SRX474501 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1332603_FANhRrbs.CG.+.wig.gz |
20.8 Mb |
(ftp)(http) |
WIG |
| GSM1332603_FANhRrbs.CG.-.wig.gz |
20.9 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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