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| Status |
Public on Jun 17, 2019 |
| Title |
iXist-Chr3_WtapKO_Dox2 |
| Sample type |
SRA |
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| Source name |
iXist-Chr3_WtapKO(mESC)
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| Organism |
Mus musculus |
| Characteristics |
strain background: 129S X Cast
gender: Male
cell line: Cell line integrating with FLXist randomly on Chr3 (P4D7F4)
cell type: mESC
agent: Dox
time course: 24h
medium: ES medium
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| Treatment protocol |
Dox treatment
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| Growth protocol |
ES cells were grown in DMEM media supplemented with 10% fetal calf serum (Seralab), 2 mM L-glutamine, 1X non-essential amino acids, 50 µM β-mercaptoethanol, 1X penicillin / streptomycin (all from Life Technologies) and 1000 U/mL LIF (made in-house). Mitomycin-inactivated mouse fibroblasts were used as feeders. All ES cells were grown in feeder-dependent conditions on gelatinised plates at 37°C with 5% CO2. Inducible Xist expression was driven by a TetOn promoter induced by addition of 1.0 to 1.5 µg/mL of doxycycline for 24 hours.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Chromatin RNA was extracted from one confluent 15 cm dish of mESCs. Briefly, cells were trypsinised and washed in PBS. Cells were lysed on ice in RLB (10 mM Tris pH 7.5, 10 mM KCl, 1.5 mM MgCl2, and 0.1% NP40), and nuclei were purified by centrifugation through a sucrose cushion (24% sucrose in RLB). The nuclei pellet was resuspended in NUN1 (20 mM Tris pH 7.5, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol), then lysed with NUN2 (20 mM Hepes pH 7.9, 300 mM, 7.5 mM MgCl2, 0.2 mM EDTA, 1 M Urea). Samples were incubated for 15 minutes on ice then centrifuged at 2800 g to isolate the insoluble chromatin fraction. The chromatin pellet was resuspended in TRIzol by passing multiple times through a 23 gauge needle. Finally chromatin-associated RNA was purified through standard TRIzol/chloroform extraction followed by isopropanol precipitation. Samples were then treated with Turbo DNAse, and 500ng – 1µg of RNA was used for library preparation using the Illumina TruSeq stranded total RNA kit. Libraries were quantified by qPCR with KAPA Library Quantification DNA standards (KAPA Biosystems). The libraries were pooled and 2X 81 paired end sequencing was performed using Illumina NextSeq 500.
TruSeq RNAseq
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Raw_Ratio_chr3.xlsx
WtapKO_DoxB
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| Data processing |
Library strategy: ChrRNA-seq
The RNA-seq data mapping pipeline was similar to our previous study (Pintacuda et al., 2017). Briefly, the raw fastq files of read pairs were first mapped to an rRNA build by bowtie2 (v2.3.2) (Langmead and Salzberg, 2012) and rRNA-mapped reads discarded. The remaining unmapped reads were aligned to the ‘N-masked’ genome (from mm10 coordinates) with STAR (v2.4.2a) using parameters “--outFilterMultimapNmax 1 --outFilterMismatchNmax 4 --alignEndsType EndToEnd” for all the sequencing libraries (Dobin et al., 2013). Unique alignments were retained for further analysis. We made use of 23,005,850 SNPs between Cast and 129S genomes and employed SNPsplit to split the alignment into distinct alleles (Cast and 129S) using the parameter “--paired”. The (allelic) read numbers were counted by the program featureCounts (-t transcript -g gene_id -s 2) (Liao et al., 2014) and the alignments were sorted by Samtools (Li et al., 2009). Bigwig files were generated by Bedtools (Quinlan and Hall, 2010) and visualized by IGV (Robinson et al., 2011) or UCSC Genome Browser. For biallelic analysis, counts were normalized to 1 million mapped read pairs (as CPM) by the edgeR R package. Genes with at least 10 SNP-covering reads across all the samples were further taken to calculate the allelic ratio of where Xi and Xa indicate inactive and active allele, respectively.
Genome_build: mm10
Supplementary_files_format_and_content: The processed data files report allelic ratio between Xi and (Xi+Xa)
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| Submission date |
Sep 06, 2018 |
| Last update date |
Jun 17, 2019 |
| Contact name |
Guifeng Wei |
| E-mail(s) |
guifeng.wei@bioch.ox.ac.uk
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| Organization name |
University of Oxford
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| Department |
Department of Biochemistry
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| Lab |
Brockdorff Lab
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3QU |
| Country |
United Kingdom |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE119602 |
Systematic Analysis of Allelic Silencing Defines the Interplay of Key Pathways in Xist-mediated Chromosome Inactivation |
| GSE119607 |
Xist-mediated Chromosome Inactivation |
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| Relations |
| BioSample |
SAMN09986221 |
| SRA |
SRX4650388 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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