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| Status |
Public on Jun 15, 2015 |
| Title |
ES_Tsix-stop_HiC |
| Sample type |
SRA |
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| Source name |
Embryonic Stem Cells (ESCs)
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| Organism |
Mus musculus |
| Characteristics |
cell type: ESCs
strain background: 129/SV-Jae x Cast
measurement: HiC
treatment: Undifferentiated in Ndiff and 2i+LIF
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| Treatment protocol |
Induction of ESC differentiation has been described by Barakat et al. (2011). In short, ESCs were split, and pre-plated on non-gelatinised cell culture dishes for 60 minutes. ESCs were then seeded in non-gelatinised bacterial culture dishes containing differentiation medium to induce embryoid body (EB) formation. EB-medium consisted of IMDM-glutamax, 15% foetal calf serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, non-essential amino acids, 37.8 μl l-1 monothioglycerol and 50 μg ml-1 ascorbic acid. EBs were plated on coverslips 1 day prior to harvesting, and allowed to grow out.
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| Growth protocol |
ESCs were cultured without feeders in the presence of leukemia inhibitory factor (LIF; 1000 U ml-1) either in Glasgow modification of Eagles medium (GMEM) containing 10% fetal calf serum (called “serum” medium), or in serum-free N2B27 supplemented with MEK inhibitor PD0325901 (1 μM), GSK3 inhibitor CH99021 (3 μM), penicillin (100 U ml-1), streptomycin (100 mg ml-1), glutamine (1 mM), non-essential amino acids (0.1 mM), β-mercaptoethanol (0.1 mM) (together called “2i” medium; Ying et al., 2008). For adaptation to 2i, serum ESCs were transferred to 2i medium and cultured for >12 days (>6 passages) in 2i medium. ESCs used in this study include the female lines ES_Tsix-stop (Luikenhuis et al., 2001), ES_Xist-del (a polymorphic 129:Cast female ESC line that show non-random XCI due to a deletion in the Xist gene on the 129 allele; Csankovszki et al. (1999)), TNGA (Chambers et al., 2007) and XT67E1 (a polymorphic 129:PGK female ESC line that show non-random XCI due to a deletion in the Xist gene on the 129 allele; Penny et al., 1996), and the male ESC lines E14Tg2a (E14) and Rex1GFPd2 lines (Wray et al., 2011; Marks et al., 2012). Derivation and culture of the EpiSCs was described previously (Maruotti et al. 2010; Veillard et al., 2014). Derivation of NPC lines, including culture conditions and further details, has been described in Splinter et al. (2011).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s recommendations. 100 μg total RNA was subjected to two rounds of poly(A) selection (Oligotex mRNA Mini Kit; QIAGEN), followed by DNaseI treatment (QIAGEN). 100–200 ng mRNA was fragmented by hydrolysis (5x fragmentation buffer: 200mM Tris acetate, pH8.2, 500mM potassium acetate and 150mM magnesium acetate) at 94°C for 90 s and purified (RNAeasy Minelute Kit; QIAGEN). cDNA was synthesized using 5 μg random hexamers by Superscript III Reverse Transcriptase (Invitrogen). Ds cDNA synthesis was performed in second strand buffer (Invitrogen) according to the manufacturer’s recommendations and purified (Minelute Reaction Cleanup Kit; QIAGEN). Strand-specific rRNA depleted ds cDNA profiling used for the NPC lines was performed with the ScriptSeq kit (cat. no. SS10924) from Illumina, according to the instructions of the manufacturer. rRNA depletion was performed with the Ribo-Zero rRNA Removal Kit using 5 μg of total RNA (Human/Mouse/Rat; cat. no. RZH110424). Collection of the cells for Hi-C and the HiC sample preparation procedure was performed as previously described (Lieberman-Aiden et al., 2009), with the slight modification that DpnII was used as restriction enzyme during initial digestion.
For the PolyA+ samples, cDNA was prepared for sequencing by end repair of 20 ng ds cDNA as measured by Qubit (Invitrogen). Adaptors were ligated to DNA fragments, followed by size selection (~300 bp) and 14 cycles of PCR amplification. Quality control of the adaptor-containing DNA libraries of both PolyA+ and ScriptSeq samples was performed by qPCR and by running the products on a Bioanalyzer (BioRad). Cluster generation and sequencing (32-42 bp) was performed with the Illumina Genome Analyzer IIx or Hi-Seq 2000 platforms according to standard Illumina protocols. For HiC, paired-end libraries were prepared according to Lieberman-Aiden et al. (2009) and sequenced on the NextSeq 500 platform using 2*75 bp sequencing.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: HiC
Initial data processing and base calling was performed using the Illumina Analysis Pipeline. FASTQ files were mapped using GSNAP version 2011-03-10 (Wu et al., 2010). To avoid bias in the mapping of either the Cast- or the 129-derived reads, the alternative alleles of polymorphic sites between the 129- and Cast-genome (see above) are included in the reference during mapping (GSNAP SNP-tolerant mapping; flag –v). Only sequence tags aligning to a single position on the genome were considered for further analysis on the 32-42 bp aligned sequence reads. Further analysis was performed with the 32-42 bp aligned sequence.
The data was converted to Browser Extensible Data (BED) files for downstream analysis. To compensate for differences in sequencing depth and mapping efficiency among samples, the total number of unique reads of each sample was uniformly equalized relative to the sample with the lowest number of sequence reads, allowing quantitative comparisons. Wiggle (WIG) files for viewing the data in the UCSC Genome Browser were generated from the normalized files.
To obtain RNA-Seq gene expression values (RPKM), we used Genomatix (www.genomatix.de; ElDorado 12-2010) selecting RefSeq genes.
Known polymorphic sites between the mouse species 129 and Cast were collected using polymorphic sites determined by (i) the Sanger mouse sequencing project using the March 2011 release (Keane et al., 2011; Yalcin et al., 2011) (http://www.sanger.ac.uk/resources/mouse/genomes/; we used the ftp-mouse.sanger.ac.uk/REL-1003/20100301-high-confidence-snps.tab file for the species 129S1, C57BL and CAST) and (ii) the NIEHS/Perlegen mouse resequencing project (Frazer et al., 2007) (http://phenome.jax.org/db/q?rtn=projects/projdet&reqprojid=198; we used the b04_Chr*_genotype.dat files for the species 129S1/SvImJ, CAST/EiJ and the C57BL/6J reference genome of NCBI Build 36 (Waterston et al., 2002)). This resulted in a total of 20,785,351 polymorphic sites between the genomes of 129 and Cast.
Within the individual samples, we used the mapped tags to determine the sequence tag coverage per allele for each of the 20,785,351 polymorphic sites using GSNAP tally. A total of 7,854,031 polymorphic sites were covered at least once in any of the samples used for this study. Per single polymorphic nucleotide, the pile-ups were subsequently assigned to either the 129- or the Cast-allele using custom Perl-based scripts (see supplementary table Polymorphic_sites_of_Tsix_stop_with_counts.txt.gz). To avoid including counts from positions which were reported to be polymorphic in the Sanger sequencing project and/or the NIEHS/Perlegen resequencing projects, but which were not present in the genotypes used for the current study, we selected polymorphic sites that were covered at least twice from both the 129 and the Cast allele. This resulted in a total of 1,121,809 polymorphic sites used in further analysis. Counts over polymorphic sites within exons of individual RefSeq genes for either 129 or Cast were summed to obtain allele-specific gene expression counts for both species. The ratio between the 129 counts or the Cast counts versus the total counts (129 + Cast) represent the relative contribution of the 129 or Cast allele, respectively, to expression of a particular gene. To calculate allele specific expression values, we multiplied the relative contribution of either 129 or Cast with the total RPKM expression value of a gene. For the ESC differentiation timecourse, only genes that contained a count of >80 over the complete timecourse from both the 129 as well as from the Cast allele were included for further analysis.
For HiC, reads were mapped to the reference mouse genome (mm9) using BWA MEM (Li et al., 2014) with default parameters. Reads were filtered based on mapping quality score (mapQ>=10) and PCR duplicates were removed. (Normalized) interaction matrices at a resolution of 40 kb and the corresponding two-dimensional heat maps were generated as previously described by Lieberman-Aiden et al. (2009) using optimized LGF normalization (normLGF) from the R package HiTC (Servant et al., 2012). The Hi-C Domain Caller package was used to calculate the directionality index from the normalized interaction matrix and to determine domains and boundaries using default parameters (Dixon et al., 2012; see Domains.txt).
Genome_build: mm9
Supplementary_files_format_and_content: Bed files were generated according to general instructions and were used for analysis; Wig files can be directly uploaded to the UCSC genome browser. The domains within Domains.txt were determined using the HiC data
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| Submission date |
Apr 07, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Hendrik Marks |
| E-mail(s) |
h.marks@ncmls.ru.nl
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| Organization name |
Radboud University Nijmegen, RIMLS
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| Department |
Molecular Biology
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| Street address |
Geert Grooteplein 26/28
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| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE60738 |
Dynamics of gene silencing during X inactivation using allele-specific RNA-Seq |
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| Relations |
| BioSample |
SAMN03464334 |
| SRA |
SRX982053 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1652666_Domains.txt.gz |
13.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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