|
| Status |
Public on May 31, 2016 |
| Title |
MBD-1 |
| Sample type |
SRA |
| |
|
| Source name |
Cortical neuronal culture, MBD precipitated
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain/background: Sprague-Dawley
cell type: cortical neuronal culture
age: embryonic day 18, 11 DIV
treatment: untreated (media alone)
mbd-capture: yes
|
| Treatment protocol |
Neurons were not treated prior to DNA extraction.
|
| Growth protocol |
Neurons were removed from cortical tissue at embryonic day 18, and maintained in vitro for 11 days prior to experimental treatment. Each culture well contained ~250,000 cells.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted, RNase-treated, and purified (DNeasy Blood and Tissue Kit, Qiagen). 2µg of total DNA was sonicated to 200-400bp and prepared for MBD-based methylated DNA capture (MethylMiner Kit, Life Technologies). MBD-IP and Input samples underwent library construction and whole-genome sequencing.
DNA sequencing was performed at Hudson Alpha using NEBNext reagents (New England Biolabs) according to manufacturer's recommendations with minor modifications (including the use of custom library adapters and indexes). DNA libraries were quantified with the Kapa Library Quant Kit (Kapa Biosystems), and underwent sequencing (25M total 50 bp single-end reads) on an Illumina sequencing platform (HiSeq 2000). We sequenced two biological replicates, as well as an input (non-IP) control for normalization.
|
| |
|
| Library strategy |
MBD-Seq |
| Library source |
genomic |
| Library selection |
MBD2 protein methyl-CpG binding domain |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Raw single-end sequenced reads (as fastq format) underwent quality control (FASTQC) in Galaxy.
Low-quality reads were filtered with the FASTX toolkit in Galaxy ("filter by quality"), with a quality cutoff of 20 and a minimum percentage of 90.
High-quality reads were aligned to the rat genome (rn5) in Galaxy using Bowtie.
Genome-aligned sequenced reads for each sample were examined using SeqMonk (Babraham institute), using Ensembl v. 70 gene and feature annotations. Due to library size, reads were extended by 250 bp to recapitulate the ~300bp fragment size average. For each sample, gene and promoter (+/-2.5kb from TSS) methylation were determined by computing the reads per million mapped reads (RPM) from MBD-IP samples.
Genome_build: Rnor_5.0 (rn5)
Supplementary_files_format_and_content: Matrix with RPM values for each sample (columns) and each gene (rows). The first row contains column identifiers. The first column contains RefSeq gene names. Promoter and gene body RPM estimates are provided in separate files.
|
| |
|
| Submission date |
Jan 14, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Jeremy Jason Day |
| E-mail(s) |
jjday@uab.edu
|
| Phone |
205-996-8960
|
| Organization name |
UAB
|
| Department |
Neurobiology
|
| Lab |
Day
|
| Street address |
1825 University Blvd
|
| City |
Birmingham |
| State/province |
AL |
| ZIP/Postal code |
35294 |
| Country |
USA |
| |
|
| Platform ID |
GPL14844 |
| Series (2) |
| GSE64987 |
Extra-coding RNAs regulate neuronal DNA methylation and long-term memory formation [MBD-seq] |
| GSE64988 |
Extra-coding RNAs regulate neuronal DNA methylation dynamics |
|
| Relations |
| BioSample |
SAMN03284616 |
| SRA |
SRX843557 |
