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Sample GSM1633772 Query DataSets for GSM1633772
Status Public on Jun 01, 2015
Title INPUT_SC_seq_mus_liver_A
Sample type SRA
 
Source name Liver A
Organism Mus musculus
Characteristics tissue: Liver
strain/cell type: C57BL/6
antibody: n/a
Growth protocol Liver samples extracted from C57BL/6 mice aged ~120 days. MEFs cultured in standard media. iPSC were dervied followign establisehd protocols outlined by Yusa et al 2013 [PMID: 24071911]
Extracted molecule genomic DNA
Extraction protocol Sheared gDNA was enriched using Active motif rabbit polyclonal against hydroxymethylation (cat#39769). prior to enrichment through magnetic IgG beads (Dynabeads protein G (Invitrogen #100-03D). 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 10 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 5hmc 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 hmc-DIP 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
 
Description mapped reads (mm10) were binned across 200bp windows.
Data processing Reads mapped to mm10 mouse build in the cloud using TMAP software
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: mm10
Supplementary_files_format_and_content: .wig file
 
Submission date Mar 13, 2015
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 (1)
GSE66889 Genome wide 5hmC patterns in the mouse liver, embryonic fibroblasts and induced pluripontent stem cells.
Relations
BioSample SAMN03408078
SRA SRX956490

Supplementary file Size Download File type/resource
GSM1633772_LiverA_INPUT_200bpBIN.wig.gz 87.3 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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