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Sample GSM3127769 Query DataSets for GSM3127769
Status Public on Jun 26, 2019
Title CBMS1_rep2_d6_EpiLC_EB_d4
Sample type SRA
Source name CBMS1_d6
Organism Mus musculus
Characteristics strain/background: CBA/MsM
cell type: day6 differentiated ESCs
Treatment protocol For differentiation, CBMS1 mESCs were differentiated to EpiLCs for 2 days and then switched to aggregation culture (EB/embryoid body culture) in Nunclon Sphera 96U-well plates (ThermoFisher, #174925), starting from 2,000 EpiLCs per well exactly as described (Hayashi K et al. Nat. Protoc., 2013), except for the use of plain GK15 medium (Hayashi K et al. Nat. Protoc., 2013) without any additional factors added during the aggregation culture. This process is practically identical to the SFEBq neural method of mESC differentiation (serum-free floating culture of EB-like aggregates with quick reaggregation) (Eiraku et al. Nat. Protoc., 2012) except that we started from EpiLCs instead of mESCs. In our hands, this resulted in efficient formation of neurectoderm cells based on gene expression after 7 days of differentiation (2 days to EpiLCs and then 5 additional days of EB culture).
Growth protocol MEFs (mouse embryonic fibroblasts) were isolated from 12.5-day-old embryos from C57BL6 mice and cultured in D-MEM supplemented with 10% FBS and penicillin/streptomycin. CBMS1 mESCs have been described (Murakami et al. Development, 2011) and were grown in 2i/LIF medium as described (Hayashi K et al., Nat. Protoc., 2013). EpiSCs (female) and their culture protocol have been described and were grown in Activin/FGF medium as described (Tesar et al. 2007).
Extracted molecule genomic DNA
Extraction protocol Hi-C experiments were performed as previously reported with some modifications (Sofueva et al., 2013). Cells were fixed with 1% formaldehyde for 10 min at room temperature. Glycine was added to 0.125 M final concentration and incubated for 5 min at room temperature. Tubes were placed on ice for 15 min. Cells were washed in ice-cold PBS by centrifuging at 400 g at 4°C. Then, 1–2 x 10^6 fixed cell pellets were used for the following steps. Cell pellets were resuspended in 500 µl of 3C lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2% NP-40) with 1x protease inhibitor cocktail (Roche, 1873580), transferred to 1.5 ml Protein LoBind Tubes (Eppendorf) and incubated on ice for 20 min. The samples were spun down for 5 min at 1000 g at 4℃. After removing the supernatants carefully, pellets were resuspended in 212.5 µl of 1.2x NEBuffer 2 with 0.3% SDS and incubated for 1 h at 37°C in the Thermomixer C3 at 950 rpm (Eppendorf). Then, 25.5 µl of 20% Triton X-100 were added to 2% final concentration and the samples were incubated for 1 h at 37°C. Next, 400 U of HindIII (NEB, R0104S) was added and the samples were incubated overnight at 37°C in the Thermomixer C3 at 950 rpm. The next day, the samples were spun down for 5 min at 4°C. After removing the supernatants carefully, pellets were resuspended in 200 µl of 1x NEBuffer 2 with 300 U of HindIII and incubated for at least 8 h at 37°C in the ThermoMixer C3 at 950 rpm. The samples were spun down for 5 min at 4°C. After removing the supernatants carefully, pellets were resuspended in 200 µl of “Fill-in Mix” (1 x NEBuffer 2, 15 µM each of dATP, dGTP, dTTP and 15 µM of Biotin-14-dCTP (Invitrogen, 19518018), 25 U of Klenow (NEB, M0210S)) and incubated for 45 min at 37°C. After spinning down for 5 min at 4°C and removing the supernatants carefully, the pellets were resuspended in 100 µl of “Ligation Mix” (1x T4 DNA ligase buffer (NEB), 6 µl of T4 DNA ligase (1 U/µl, Invitrogen, 15224017)) and incubated overnight at 16°C. The sample volumes were brought up to ~300 µl by adding 10 mM Tris-HCl (pH 7.5) and RNaseA-treated for 30 min at 37°C by adding 3 µl of 10 mg/ml RNaseA. Reverse-crosslinking and proteinase K treatment were performed overnight at 65°C by adding 6.25 µl of 20 mg/ml Proteinase K. The next day, 6.25 µl of 20 mg/ml Proteinase K was added and incubated for 2 h at 65°C. Samples were then extracted twice with phenol/chloroform and once with chloroform. After this, 30 µl of 3 M sodium acetate (pH 5.2) was added and 825 µl of ice-cold EtOH was added. The samples were incubated overnight at –80°C and spun down for 30 min at 20,000 g at 4°C for DNA precipitation. After two 70% EtOH washes (add 1 ml, cfg for 20 min at 20,000 g at 4°C), the DNA pellets were resuspended in 30 µl of 10 mM Tris-HCl (pH 7.5).
For generating next-generation sequencing libraries, 2.5 µg of Hi-C DNA was used per library. To remove biotin from unligated ends, Hi-C DNA was mixed with 50 µl of “T4 DNA pol mix” (1x NEBuffer 2, 1x BSA, 100 µM dATP, 100 µM dGTP, 2.5 U of T4 DNA polymerase (NEB, M0203S)) and incubated for 2 h at 12°C. The sample volumes were brought up to 100 µl by adding 10 mM Tris-HCl (pH 7.5). Samples were extracted once each with phenol/chloroform and chloroform. After EtOH precipitation, the DNA pellets were resuspended in 130 µl of nuclease-free water. DNA samples were sheared (300–500 bp) using the following settings in Covaris S220 (Duty Factor: 5.0, Peak incident power: 175 W, Cycles per burst: 200, Treatment time: 120 sec) using microTUBE with snap cap (Covaris, 520045). After DNA shearing, DNA size selection was performed using AMPure XP beads (Beckman Coulter). Using the size-selected DNA samples, biotin pull-down and sequencing library preparations were performed using 30 µl of Dynabeads MyOne Streptavidin C1 (Invitrogen, 65001) and a part of Nextera Mate Pair Sample Preparation kit (Illumina, FC-132-1001) following the kit’s protocol at half scale. For library amplification, KAPA Real-Time Amplification Kits (KAPA BIOSYSTEMS, KK2701) were used to determine the optimal PCR cycles (9–12 cycles) and the final PCR products were purified by AMPure XP beads.
Library strategy Hi-C
Library source genomic
Library selection other
Instrument model Illumina HiSeq 1500
Description Biological replicate 2 of 2.
Data processing Read pairs were individually mapped to the mouse genome (UCSC mm9) using the hiclib pipeline (Imakaev et al., 2012) with iterative mapping method.
After read mapping, each side of the mapped reads was also applied to the hiclib pipeline. First, uniquely mapped paired-reads were assigned to HindIII fragments. Only those with correct orientations were considered valid pairs, and non-ligated and self-ligated read pairs were filtered out. Fragment-level filters were next applied to remove invalid reads: reads that started within 5 bp from the restriction sites, duplicated read pairs, extremely large and small restriction fragments (> 100 kbp and < 100 bp, respectively), and extremely high and low count restriction fragments (top 0.5% of all counts and zero counts, respectively).
Biological replicates were merged, and Hi-C contact heatmaps were generated in 200-kb or 40-kb non-overlapping genomic bins for both merged samples (mainly used for visualization) and for single biological replicates.
To correct the bias of the contact heatmaps, iterative correction was performed with the following steps: diagonal removal was applied to only the first diagonal (“removeDiagonal(0)”), bin removal was done by the sequence fraction (0.5), and finally high trans contacts (the fraction of the top 0.05% trans contacts) were removed. The bias-corrected contact heatmaps were used to generate the A/B compartment profiles (200-kb bins or 200-kb sliding windows at 40-kb interval) in each chromosome as previously described (Lieberman-Aiden et al., 2009) by the hiclib pipeline with a small modification in the ∆“doCisPCADomains(domaidunction=”lieverman-”)” function including observed / expected analysis, covariance matrix generation from the Pearson correlation matrix and principal component analysis. The first eigenvalues were used to identify A/B compartments (i.e. Hi-C PC1). Because positive and negative eigenvalues are arbitrary, correlation with the GC content was used to assign positive eigenvalues as ‘A compartment’ and negative eigenvalues as ‘B compartment’. After obtaining A/B compartment profile, quantile normalization was performed using the limma package (Ritchie et al., 2015).
Genome_build: UCSC mm9 (MGSCv37)
Supplementary_files_format_and_content: bedGraph
Submission date May 02, 2018
Last update date Jun 27, 2019
Contact name Ichiro Hiratani
Phone +81-78-306-3179
Organization name RIKEN
Department Center for Developmental Biology
Lab Laboratory for Developmental Epigenetics
Street address 2-2-3 Minatojima-minamimachi, Chuo-ku
City Kobe
State/province Hyogo
ZIP/Postal code 650-0047
Country Japan
Platform ID GPL18480
Series (2)
GSE113981 Single-cell DNA replication profiling identifies spatiotemporal developmental dynamics of chromosome organization [HiC-seq]
GSE113985 Single-cell DNA replication profiling identifies spatiotemporal developmental dynamics of chromosome organization
BioSample SAMN09015366
SRA SRX4024414

Supplementary file Size Download File type/resource
GSM3127769_CBMS1_rep2_d6_EpiLC_EB_d4.w200k.bedGraph.gz 148.1 Kb (ftp)(http) BEDGRAPH
GSM3127769_CBMS1_rep2_d6_EpiLC_EB_d4.w200ks40k.bedGraph.gz 762.6 Kb (ftp)(http) BEDGRAPH
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Raw data are available in SRA
Processed data provided as supplementary file

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