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Sample GSM1552786 Query DataSets for GSM1552786
Status Public on May 06, 2015
Title rsEpiSCs Rep #2 [methylC-Seq]
Sample type SRA
 
Source name Region selective epiblast stem cells
Organism Mus musculus
Characteristics cell type: E5.75 derived rsEpiSCs
passages: 20-25
strain/variation: CR x Oct4 PE-GFP transgenic
Extracted molecule genomic DNA
Extraction protocol One µg of genomic DNA was spiked with 5 ng unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI). The DNA was fragmented with a Covaris S2 (Covaris, Woburn, MA) to 150-200 bp, followed by end repair and addition of a 3’ A base. Cytosine-methylated adapters provided by Illumina (Illumina, San Diego, CA) were ligated to the sonicated DNA at 16˚C for 16 hours with T4 DNA ligase (New England Biolabs). Adapter-ligated DNA was isolated by two rounds of purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA). Adapter-ligated DNA (≤450 ng) was subjected to sodium bisulfite conversion using the MethylCode kit (Life Technologies, Carlsbad, CA) as per manufacturer’s instructions. The bisulfite-converted, adapter-ligated DNA molecules were enriched by 8 cycles of PCR with the following reaction composition: 25 µL of Kapa HiFi Hotstart Uracil+ Readymix (Kapa Biosystems, Woburn, MA) and 5 µl TruSeq PCR Primer Mix (Illumina) (50 µl final). The thermocycling parameters were: 95˚C 2 min, 98˚C 30 sec, then 4 cycles of 98˚C 15 sec, 60˚C 30 sec and 72˚C 4 min, ending with one 72˚C 10 min step. The reaction products were purified using AMPure XP beads. Up to two separate PCR reactions were performed on subsets of the adapter-ligated, bisulfite-converted DNA, yielding up to two independent libraries from the same biological sample.
 
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina HiSeq 2500
 
Data processing Sequencing reads were first trimmed for adapter sequence using Cutadapt.
All cytosines in the trimmed reads were then computationally converted to thymines and mapped twice, to a converted forward strand reference and to a converted reverse strand reference both based on the mm10 reference genome.
A converted reference is created by replacing all cytosines with thymines (forward strand) or all guanines with adenines (reverse strand) in the reference FASTA file.
For mapping, we used Bowtie3 with the following options: "-S","-k 1","-m 1","--chunkmbs 3072","--best","--strata","-o 4","-e 80","-l 20", and "-n 0".
Any read that mapped to multiple locations was removed and one read from each starting location on each strand from each library was kept (i.e., clonal reads were removed).
Genome_build: GRCm38 (mm10)
Supplementary_files_format_and_content: The processed data for the allc_<sample>_<chr>.tsv files contain a row of headers indicating the chromosome, position, strand, methylation class, methylation count, total coverage, and methylation call. These are tab-delimited files.
 
Submission date Nov 21, 2014
Last update date May 15, 2019
Contact name Christopher Benner
E-mail(s) cbenner@ucsd.edu
Organization name University of California, San Diego (UCSD)
Department Medicine
Street address 9500 Gilman Dr. MC 0640
City La Jolla
State/province California
ZIP/Postal code 92093-0640
Country USA
 
Platform ID GPL17021
Series (2)
GSE60605 An alternative pluripotent state confers interspecies chimaeric competency
GSE63568 An alternative pluripotent state confers interspecies chimaeric competency [WGBS]
Relations
BioSample SAMN03216800
SRA SRX765922

Supplementary file Size Download File type/resource
GSM1552786_allc_LP_EpiSC_2.tsv.gz 3.9 Gb (ftp)(http) TSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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