|
| Status |
Public on May 17, 2016 |
| Title |
WT1_liver_5hmC_input [hmeDIP-seq] |
| Sample type |
SRA |
| |
|
| Source name |
WT Liver Tissue
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6J
genotype/variation: wild type
Sex: male
tissue type: liver
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sheared gDNA was enriched using Anti 5hmc - Active Motif prior to enrichment through magnetic IgG beads. Following a series of buffer washes the DNA is then released through proteinase K digestion of the antibody and the DNA is subsequently cleaned up using Qiagen QIAquick PCR purification kits (Qiagen) and eluting in 20ul purified water. Enriched DNA was then subjectedto 15 cycles of whole genome amplification using using an enhanced amplification kit optimised for next generational sequencing (Sigma-Aldrich SeqPlex DNA Amplification Kit).
Individual libraries were generated from 100ng each of 5mc enriched and input DNA for each sample, using the Ion XpressPlus Fragment Library Kit (life Technologies™). The DNA was end repaired, purified and then ligated to Ion-compatible barcoded adapters (Ion Xpress™ Barcode Adapters 1–96: Life Technologies™), followed by nick-repair to complete the linkage between adapters and DNA inserts. The adapter-ligated library was then amplified (10 cycles) and finally size-selected using two rounds of AMPure XP bead capture to size‐select fragments approximately 100–250 bp in length. An equimolar pool of barcoded libraries was prepared at 100pM; each pool contained an meDIP enriched and corresponding input sample. 8pM of the pooled library was added into an emulsion PCR based template reaction; in this reaction the fragments generated during the library prep were attached to Ion sphere particles (ISPs) and clonally amplified. This process was carried out using the Ion One Touch 2 system and the Ion P1 Template OT2 200 Kit.
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Ion Torrent Proton |
| |
|
| Data processing |
Reads mapped to mm10 mouse build
Data binned into 200bp windows across the genome
Data converted into mm9 build and into a wiggle (.wig) file
data normalised by read count
enriched (5hmC) datasets normalised to matched input dataset to generate enrichment over background dataset
Genome_build: mm9
Supplementary_files_format_and_content: .wig file
|
| |
|
| Submission date |
Feb 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
John Paterson Thomson |
| E-mail(s) |
john.thomson@igmm.ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Department |
MRC Human Genetics Unit
|
| Lab |
Meehan
|
| Street address |
Crewe Road
|
| City |
Edinburgh |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL18635 |
| Series (2) |
| GSE77730 |
DNA IP for 5hmC modified CpGs in the liver of control mice and a Tet1 -/- mouse liver [hmeDIP-seq] |
| GSE77731 |
Profiling of epigenetic and transcriptomic landscapes in normal mouse liver, phenobarbital exposed mouse livers and mouse liver tumours |
|
| Relations |
| BioSample |
SAMN04481704 |
| SRA |
SRX1568344 |
