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Sample GSM5105942 Query DataSets for GSM5105942
Status Public on Feb 25, 2021
Title hMeDIP-seq-OL-2
Sample type SRA
Source name OL culture
Organism Mus musculus
Characteristics strain stage: Differentiated OPC
strain name: Tet1fl/+;Olig1Cre/+
Growth protocol Mouse or rat OPCs were isolated from P6 cortices of mice by immunopanning with antibodies against Ran-2, GalC and O4 sequentially. Briefly, cerebral hemispheres were diced and digested with papain at 37°C. Following gentle trituration, cells were resuspended in a panning buffer containing insulin (5 µg/ml) and then incubated at room temperature sequentially on three immunopanning dishes: Ran-2, anti-GalC, and O4. O4+GalC− OPCs were released from the final panning dish with trypsin. To induce the differentiation of OPCs, mitogens in the culture medium (FGF and PDGFaa) were replaced with T3, NAC and CNTF.
Extracted molecule genomic DNA
Extraction protocol For hMeDIP-seq, genomic DNA was sonicated to ~200-800bp with a Bioruptor sonicator (Diagenode). 800 ng of sonicated DNA was end-repaired, A-tailed, and ligated to single-end adapters following the standard Illumina genomic DNA protocol (FC-102-1002, Illumina). After agarose size-selection to remove unligated adapters, the adaptor-ligated DNA was used for immunoprecipitation (IP) with a mouse monoclonal anti-5-hydroxymethylcytosine antibody (Diagenode, C15200200). For this, DNA was heat-denatured at 94°C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 μL primary antibody overnight at 4°C with rocking agitation in 400 μL IP buffer (0.5% BSA in PBS). To recover the immunoprecipitated DNA fragments, 20 μL of magnetic beads were added and incubated for an additional 2 hours at 4°C with agitation. After IP, a total of five washes were performed with ice-cold IP buffer. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg/mL proteinase K for 2 hours at 65°C and then allowed to cool down to room temperature. DNA was then purified using Qiagen MinElute columns and eluted in 16 μL EB (Qiagen).
14 cycles of PCR were performed on 5 μL of the immunoprecipitated DNA using the single-end Illumina PCR primers. The resulting products were purified with Qiagen MinElute columns, after which a final size selection (300-1,000 bp) was performed by electrophoresis in 2% agarose. Libraries were quality controlled by Agilent 2100 Bioanalyzer.
Library strategy MeDIP-Seq
Library source genomic
Library selection 5-methylcytidine antibody
Instrument model Illumina HiSeq 2500
Description hMeDIP-seq
Data processing Illumina Casava software used for basecalling.
Sequenced reads were trimmed for adaptor sequence.
After passing Solexa CHASTITY quality filter, the clean reads were aligned to Mus_musculus genome (UCSC MM10) using BOWTIE software (V2.1.0).
Aligned reads were used for peak calling, both mRNA and LncRNA associated hMeDIP enriched regions (peaks) with statistically significant were identified for each sample, using a q-value threshold of 10-4 by MACS v2.
wig files were generated by MACS14
Submission date Feb 24, 2021
Last update date Feb 26, 2021
Contact name Richard Lu
Organization name Cincinnati Children's Hospital Medical Center
Department CBDI
Lab Lu Lab,T6.525
Street address 3333 Burnet Ave
City Cincinnati
State/province OH
ZIP/Postal code 45229
Country USA
Platform ID GPL17021
Series (1)
GSE122838 Tet1-mediated Epigenetic Programming Regulates Calcium Homeostasis and Stage-specific CNS Myelination and Regeneration
BioSample SAMN18053385
SRA SRX10165825

Supplementary file Size Download File type/resource
GSM5105942_OL2_Mus_musculus_MM10_hisat2_alignment.bed.gz 341.5 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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