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Sample GSM1182881 Query DataSets for GSM1182881
Status Public on Nov 04, 2013
Title dRF25: 8h.LNA-C1 Xist CHART
Sample type SRA
Source name 8h.LNA-C1 Xist CHART
Organism Mus musculus
Characteristics strain: F1: 129S1/CastEiJ x 129S1
cell type: immortalized MEFs
capture oligos: C-11
sequenced molecule: Xist CHART enriched DNA
Treatment protocol d0 ES cells were cultured in the presence of LIF under adherent conditions. d7 ES cells were cultured without LIF under non-adherent conditions for 4 days and plated for an additional 3 days.
Growth protocol Cells were cultured in regular fibroblast medium (1% Pen/Strep, 1% L-glutamine, high-glucose DMEM) with either 15% FBS (MEFs) or 30% FBS (ES cells)
Extracted molecule genomic DNA
Extraction protocol Nuclei, purified from cells crosslinked in 1% formaldehyde for 10 min, were further crosslinked with 3% formaldehyde for 30 min at room temperature. Sheared chromatin, with a median size around 3kb, was incubated overnight with a mixture of biotinylated capture oligonucleotides that were complementary to Xist RNA. The hybridized material was captured by streptavidin beads, and washed extensively before RNaseH elution. Enriched DNA was purified after formaldehyde crosslink reversal and subjected to library construction.
Genome DNA of inputs and Xist CHART enriched DNA samples were fragmented by sonication, and the sequencing libraries was constructed by first repairing DNA-ends, A-tailing, ligating to universal adapters, and amplifying for 12 cycles with indexed primer.
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2500
Description 8 hr post tranfection of LNA-C1
Data processing Library strategy: CHART-Seq
Basecalls performed using Illumina software, version 1.8, except for dSP20b, which was performed using version 1.3
CHART-seq: Alignment was performed with Novoalign 3.00 ( using default parameters with modifications: -t 180 -h 180 180 -v 180. Uniquely aligned pairs were selected, discarding duplicate fragments. SPP was used to normalize each sample to its corresponding input accepting all tags and smoothing with 1000 kb windows sampled every 500bp. To normalize across data sets, each bedGraph was normalized to sum of all positive values on chr4.
RNA-seq: Adapter sequences were stripped from reads and identical reads discarded. Alignment was performed using tophat2 with the “b2-sensitive” preset and otherwise default parameters. After removal of PCR duplicates, all unique reads mapping to gene bodies were summed up for the cas, mus and comp tracks. Read numbers over genes in the allelic tracks were used to calculate skew (mus-cas/mus+cas) and genes skewed significantly (P < 0.01, cumulative binomial probability) in d7 ES cells and MEFs output to bedGraph.
Each dataset was aligned to variant CAST/EiJ and 129S1/SvJm genomes constructed using high quality polymorphisms to the C57/Bl6 reference genome (mm9 build). Pairs aligning to only one variant genome and pairs aligning better to one variant genome (in number of nucleotide edits to reference) than the other were retained (allele-specific), as were pairs aligning equally well (non-allele-specific).
Repetitive alignments, duplicate reads and low quality reads were removed. The resulting files were analyzed using SPP software. In this analysis all tags were included and the coverage generated with smoothing using 1kb bins every 500 bp to generate input-subtracted, normalized read densities. To account for different read depths across data sets, each coverage file was scaled using the total positive read density on an autosome (chr4) from the corresponding composite track (i.e., the mus, cas and comp tracks were all scaled using the same factor) and output to bedGraph.
CHIP-seq: Data from GSE36905 was aligned and processed as CHART-seq samples. Resulting coverage tracks (EZH2/K27me3) are linked directly to GSE48649 (bedGraphs).
Genome_build: mm9
Supplementary_files_format_and_content: All processed data files are in bedGraph format and represent either spp enrichment score for CHART-seq and CHIP-seq or skewed expression value ranging from -1 (all CAST/EiJ) to +1 (all 129S1) for RNA-seq
Submission date Jul 09, 2013
Last update date May 15, 2019
Contact name Stefan F Pinter
Organization name University of Connecticut, UConn Health
Department Genetics and Genome Sciences
Lab Pinter lab
Street address 263 Farmington Avenue
City Farmington
State/province CT
ZIP/Postal code 06030-6403
Country USA
Platform ID GPL17021
Series (1)
GSE48649 High-resolution chromosomal maps of Xist RNA reveal a two-step spreading mechanism during X-inactivation
BioSample SAMN02230227
SRA SRX319119

Supplementary file Size Download File type/resource
GSM1182881_8h.LNA-C1.comp.bedGraph.gz 44.6 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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