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| Status |
Public on Dec 13, 2018 |
| Title |
CBMS1_ESC_single_MidS_16 |
| Sample type |
SRA |
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| Source name |
CBMS1 ESC
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| Organism |
Mus musculus |
| Characteristics |
strain: CBA/MsM
cell type: embryonic stem cell
sample state: Single cell
xx or xo chromosomes in single cell samples: XX
xa allele (based on rt, only for cbms1 single-cell mids xx samples): CBA/MsM
xi allele (based on rt, only for cbms1 single-cell mids xx samples): NA
repliscore of 40–70%: TRUE
cell cycle phase: MidS
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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 |
hTERT-RPE1 cells were grown in MEM-alpha 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).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For BrdU-IP samples, we followed our routine protocol as described (Ryba et al., Nat. Protoc. 2011). A Sony SH800 cell sorter was used in the ultra-purity mode, fractionating early and late S-phase populations. The BrdU-IP protocol has been described in detail (Ryba et al., Nat. Protoc. 2011), except that we used a Bioruptor UCD-250 (Sonic Bio) for gDNA sonication in high output mode, with ON/OFF pulse times of 30 sec/30 sec for 6 minutes. After BrdU-IP, immunoprecipitated DNA samples were subject to WGA with a SeqPlex kit (Sigma, SEQXE). For early S-phase population samples, 200,000 cells from the first half of S-phase were sorted by Sony SH800 cell sorter, and gDNA was isolated using a Qiagen kit (Qiagen #69504, DNeasy Blood & Tissue Kit) and fragmented to 200–300 bp with a Covaris ultrasonciator (model: S220, tube: microTUBE snap-cap) according to the manufacturer’s instructions (peak incident power: 175, duty factor: 10%, cycles per burst: 200, treatment time: 120), followed by cleanup and size selection via Agencourt AMPure XP SPRI beads (Beckman Coulter). For single or 100 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). For RNA-seq samples, cells were lysed in TRI Reagent (Molecular Research Center, Inc. cat. TR 118) to extract total RNA.
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, 84, 127-bp length, single-read or paired-end read). For NGS on Ion Proton system, libraries were constructed using Ion Plus Core Module for AB Library Builder System (Thermo Fisher Scientific, #4477683) with Ion Xpress Barcode Adapters 1-16 Kit (Thermo Fisher Scientific, #4477683). For RNA-seq, NGS library preparation was performed using 500ng of total RNA following the standard protocol of TruSeq Stranded mRNA Sample Prep Kit (Illumina).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
BrdUIP, Population early-S, 100-cells, Single-cell data: 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.
BrdUIP, Population early-S, 100-cells, Single-cell data: trimmed reads were mapped into mm9, hg19, CBMS1-specific (CBA/MsM) diploid genome based on mm9 genome by 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 or hg19 were removed by using picard tool and the reads overlapped with mm9, hg19 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.
BrdUIP data: we counted the reads of early and late S-phase BrdU-IP samples in sliding windows of 200 kb at 40-kb or 80-kb intervals (written in the processed data file as “w200sk40k” or “w200ks80k”, respectively) in non-overlapping 400-kb windows (“400k”) and performed rpm (reads per million) normalization. Then, the ratio of early-S to total read counts [(Early-S reads)/(Early-S reads + Late-S reads)] was calculated for each bin and their distribution converted to fit within a ± 1 scale, and this value was defined as the BrdU-IP RT score of each bin. We filtered out bins whose total read counts were within the bottom 5% of all bins.
Population early-S data: we counted the reads of early-S and G1 in sliding windows of 200 kb at 40-kb intervals, performed rpm normalization in a manner identical to BrdU-IP data processing, and defined Log2[(Early-S reads)/(G1 reads)] as the population early-S RT score.
100-cells data: we counted the reads of 100 mid-S and G1 cells in sliding windows of 200 kb at 40-kb intervals and used the correctMappability command in the R package AneuFinder (http://bioconductor.org/packages/release/bioc/html/AneuFinder.html) for normalizing mid-S data based on G1 data. From the mappability corrected mid-S read counts, the genome-wide median was obtained and used to generate Log2[(Mappability corrected Mid-S reads)/median] scores, which we defined as the 100-cell mid-S RT score.
Single-cell data (hTERT-RPE1): For the analysis of single mid-S cells, we counted the reads in sliding windows of 200 kb at 40-kb intervals, and used the AneuFinder’s correctMappability command sfor normalizing mid-S data based on the merged G1 control. From the mappability corrected mid-S read counts, the genome-wide median was obtained and was used to generate Log2[(Mappability corrected Mid-S reads)/median] scores, which we defined as the single-cell mid-S RT score.
Single-cell data (CBMS1 un-resolved): 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 data: 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" ].
Binarized unresolved data: Sample_Name_80k_100S_MAP_2HMM2_eps0.01.binary.bedGraph, Binarized CBA data: Sample_name_cba_400k_100S_MAP_HMM2_eps0.01.binary.bedGraph, Binarized MsM data: Sample_name_msm_400k_100S_MAP_HMM2_eps0.01.binary.bedGraph
For paired-end reads, only "R1" (Fwd) reads were used for the analysis.
RNA-seq: allele-specific RNA-seq analysis was performed using CBA/MsM diploid genome as described in Sakata et al. Development 2017. We defined genes with allelic expression imbalance as those with the percentage of either MsM- or CBA-specific reads above 60% (or >70%) among genes that show ≥10 total reads (i.e. CBA+MsM read numbers combined) and >0.1 FPKM (fragments per kilobase of exon per million fragments mapped, in a non-allelic manner) in three biological replicates (CBMS1_allele_specific_genetable.txt).
Genome_build: hg19 / mm9 / CBMS1 diploid genome
Supplementary_files_format_and_content: bedGraph, bigWig
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| Submission date |
Dec 27, 2017 |
| Last update date |
Dec 19, 2018 |
| 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 |
GPL18480 |
| Series (1) |
| GSE108556 |
Genome-wide stability of DNA replication program in single mammalian cells |
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| Relations |
| BioSample |
SAMN08245351 |
| SRA |
SRX3581576 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2904998_P263_26_1_80k_100S_MAP_2HMM2_eps0.01.binary.bedGraph.gz |
131.4 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM2904998_P263_26_1_cba_400k_100S_MAP_HMM2_eps0.01.binary.bedGraph.gz |
23.7 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM2904998_P263_26_1_msm_400k_100S_MAP_HMM2_eps0.01.binary.bedGraph.gz |
23.6 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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