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| Status |
Public on Jun 20, 2020 |
| Title |
CBMS1_Day3Diff_ESC_single_G1_06 |
| Sample type |
SRA |
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| Source name |
Day3 Differentiated CBMS1 ESC
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| Organism |
Mus musculus |
| Characteristics |
strain: CBA/MsM
cell type: day3 differentiated ESCs
sample state: Single cell
xx or xo chromosomes in single cell samples: NA
xa allele (based on rt, only for cbms1 single-cell mids xx samples): NA
xi allele (based on rt, only for cbms1 single-cell mids xx samples): NA
repliscore of 40–70%: NA
cell cycle phase: G1
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| 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).
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| Growth protocol |
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).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For single cells samples, cells were sorted with a Sony SH800 cell sorter using the single-cell mode. Sample preparations were based on the previous study (Baslan et al., Genome Res. 2015). Extracted gDNA samples were subjected to WGA with a SeqPlex kit (Sigma, SEQXE). Amplified gDNA was purified and size-selected with Agencourt AMPure XP SPRI beads and the SEQXE adapter sequence was removed by the primer removal enzyme Eco57I (Sigma, SEQXE). The DNA fragment size peak should be within 150–200 bp, which was confirmed by a capillary electrophoresis system, MultiNA (Shimadzu).
NGS libraries were constructed with a NGS LTP Library Preparation Kit (KAPA, KK8232) according to the manufacturer’s instructions, with slight modifications based on the previous study (Kadota et al., Sci. Rep. 2017). For a multi-plex NGS run, a SeqCap adapter kit A/B (Roche, 07141530001/ 07141548001) and NEXTflex DNA barcode (Bio Scientific, NOVA) were used. The samples were subjected to NGS on an Illumina Hiseq 1500 system (80-bp length, single-read).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Description |
80kb resolution data
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| Data processing |
Sequence reads were trimmed to remove adapter sequences using the cutadapt program before mapping. For WGA samples, we performed a two-step adapter trimming, first removing the Illumina adapter based on the index of each NGS library and then removing the SEQXE adapter. As the SEQXE adapter sequence was not available, we empirically estimated it as the sequence that repeatedly appeared near the 5’ end.
Trimmed reads were mapped into mm9 and CBMS1-specific (CBA/MsM) diploid genome based on mm9 genomeby using bwa (ver: 0.7.10-r789, command: bwa aln => bwa samse). CBMS1-specific diploid genome was constructed as described in Sakata et al. Development (2017) with minor modifications using CBA or MsM strain genomic reads. Duplicated reads mapped into mm9 were removed by using picard tool and the reads overlapped with mm9 black list regions (https://sites.google.com/site/anshulkundaje/projects/blacklists) were filtered out. The mapped reads with MAQP>10 were used for the further analysis. For the diploid genome mapping, we defined MAPQ>16 as the allele specific reads and used the liftover tool (UCSC Genome Browser) to convert to the mm9 genome coordinates. After this, duplicate reads identified as an identical chromosome start position and strand information were filtered out.
Single-cell data (CBMS1, differentiated CBMS1, EpiSC un-resolved at 80-kb): first, we counted the reads in non-overlapping 80-kb windows, and used the AneuFinder’s correctMappability command for normalizing mid-S data (Mappability corrected Mid-S reads) based on the merged G1 control. Binarization was performed using the Mappability corrected 25per-S (25% S-phase) , Mid-S (50% S-phase), 75per-S (75% S-phase) reads described above by using the findCNVs command in AneuFinder [2-HMM; options for 25per-S: method="HMM", max.iter=3000, states=c("zero-inflation", "0-somy", "1-somy", "2-somy"), eps=0.01, most.frequent.state="1-somy"; options for Mid-S, 75per-S: method="HMM", max.iter=3000, states=c("zero-inflation", "0-somy", "1-somy", "2-somy"), eps=0.01, most.frequent.state="2-somy"; 1-somy, unreplicated; 2-somy, replicated]. In the processed data files, replicated bin was shown as 1, unrelicated bin was shown as -1, and no-data was shown as 0. “Repliscore” was calculated as a percentage of replicated bins in the total number of replicated and unreplicated bins.
Single-cell haplotype-resolved chrX data (d0, d2, d7): after the specific filtering steps for haplotype-resolved data, we counted the reads in non-overlapping 400-kb windows, and used the AneuFinder’s correctMappability command for normalizing mid-S data (Mappability corrected Mid-S reads) based on the filtered merged G1 control. Binarization was performed as described above by using the findCNVs command in AneuFinder [2-HMM; options for 25per-S: method="HMM", max.iter=5000, states=c("zero-inflation", "0-somy", "1-somy", "2-somy"), eps=0.01, most.frequent.state="1-somy" ; options for Mid-S, 75per-S: method="HMM", max.iter=5000, states=c("zero-inflation", "0-somy", "1-somy", "2-somy"), eps=0.01, most.frequent.state="2-somy"].
For paired-end reads, only "R1" (Fwd) reads were used for the analysis.
Binarized 40kb data: Sample_Name_40k_100S_MAP_2HMM2_eps0.01.binary.bedGraph
Genome_build: mm9
Supplementary_files_format_and_content: We provide three types of processed data files. One is the binarized data (scRepliseq_data_884cells_G1_Sphase_binary_data_80kb.txt). Second is the sample information data (scRepliseq_data_sample_Info.txt). Third is the haplotype-resolved Mid-S binarized data of chrX (scRepliseq_d0_33_CBA_XX_chrX_only_400kb.txt, scRepliseq_d0_33_MsM_XX_chrX_only_400kb.txt, scRepliseq_d2_27_CBA_XX_chrX_only_400kb.txt, scRepliseq_d2_27_MsM_XX_chrX_only_400kb.txt, scRepliseq_d7_36_XX_Xa_chrX_only_400kb.txt, scRepliseq_d7_36_XX_Xi_chrX_only_400kb.txt). We also provide README file for the description of each processed file.
Supplementary_files_format_and_content: bedGraph
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| Submission date |
Jun 19, 2020 |
| Last update date |
Jun 21, 2020 |
| Contact name |
Ichiro Hiratani |
| E-mail(s) |
ichiro.hiratani@riken.jp
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| Phone |
+81-78-306-3179
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| Organization name |
RIKEN
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| Department |
Center for Developmental Biology
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| Lab |
Laboratory for Developmental Epigenetics
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| Street address |
2-2-3 Minatojima-minamimachi, Chuo-ku
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| City |
Kobe |
| State/province |
Hyogo |
| ZIP/Postal code |
650-0047 |
| Country |
Japan |
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| Platform ID |
GPL21273 |
| Series (2) |
| GSE113985 |
Single-cell DNA replication profiling identifies spatiotemporal developmental dynamics of chromosome organization |
| GSE114470 |
Single-cell DNA replication profiling identifies spatiotemporal developmental dynamics of chromosome organization [scRepli-seq] |
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| Relations |
| BioSample |
SAMN15330492 |
| SRA |
SRX8586778 |
| 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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