Randomization and statistical analysis of ad hoc data from groups and genotypes in GraphPad Prism v.9.5.0 with an application to comparisons with multiple replicated experiments

All experiments were replicated at least twice with the same conclusion. Each figure legend states the exact numbers of replicated experiments. Samples and animals were randomly allocated to experimental groups and proceeded in experiments, and data were collected and analysed blind and ad hoc registered to groups or genotypes.

GraphPad Prism v.9.5.0 was used for statistical analysis. Mean and s.e.m. were used to represent a sample. Two samples were compared with multiple data points using Chi-squared and twotailed Student’s t-tests. No statistical methods were used to predetermine sample sizes.

Quantifying the size of adipocytes and droplets in iBAT using the Fiji method: a comparative study with a digital slide scanner

The size of the adipocytes in iBAT was quantified using the Fiji method. In brief, WAT and iBAT were processed to paraffin-embedded sections (3 μm), stained with haematoxylin and eosin and scanned into digital images using a NanoZoomer-SQ Digital slide scanner (Hamamatsu). Images were randomly picked and the plug-in Analyse Particles was used to count the number and measure the size of adipocytes and droplets in iBAT. 11,000 droplets were measured for each iBAT.

The percentages of PDGFRα+ and DES+ cells were counted manually based on PDGFRα and DES signals. The percentages of TH+ and NPY+ neurons in the hypothalamus and ventral tegmental area were also counted manually. The views from 531, 531 and 30m were projected to the audience using a script. Labelled areas were segmented using a threshold set by default, and coverage was calculated as area NPY+/area CD31+.

The percentage of EdU+ cells was counted using a tool that detected particles. Threshold was set automatically using the Otsu thresholding method.

The surface tool was used to quantify the innervation of NPY+ axons. Labelled areas of NPY+ axons and CD31+ vessels in whole cleared iWAT were automatically segmented, and the innervation of NPY in vasculature was calculated as volumeNPY+/volumeCD31+. The coverage of DES+ mural cells in iWAT was calculated similarly, as volumeDES+/volumeCD31+. Using an automatic unbiased method, confocal images showing NPY+ innervation were quantified. AreaNPY+ and areaCD31+ were segmented using the Otsu thresholding method and measured with the ‘measure’ program.

Overlapping between NPY and CD31 was calculated using a plug-in. There are randomly picked regions that are projected to the right using a script. The boundaries were automatically categorized using a threshold that was set by default.

Source: Sympathetic neuropeptide Y protects from obesity by sustaining thermogenic fat

Analysis of GEO datasets for cell proliferation studies using the Click-iT EdU Cell Proliferation Kit (ThermoFisher)

Public scRNA-seq datasets were downloaded from Gene Expression Omnibus (GEO) and analysed using the following method. Cells with fewer than 200 unique detected genes or over 5% mitochondrial counts were discarded. After filtering, the gene x cell matrix was normalized using ‘NormalizeData()’ in Seurat v.4.2.0 (ref. 50) in R (v.4.2.2). In addition to the data being scaled using ‘ScaleData,’ linear dimensional reduction and calculations of UMAP coordinates for all cells were also performed. Cells with dimensions set to 15 were clustered using the search tool’FindClusters()+’. Each cluster was identified based on differentially expressed genes and known markers in the published literature5,22,29,51,52,53.

The cell proliferation assay was performed with the Click-iT EdU Cell Proliferation Kit (ThermoFisher, catalogue no. C10340) for imaging. Mural cells were seeded at 0.5 × 105 per millilitre in 12-well plates on coverslips and cultured overnight before experiments. Cells were cultured with either 1 M NPY or 2 M. For 6 hours in medium for mural cells, the Cell Signaling catalogue no. 99000 S says PD98059. Cells were fixed with 4% PFA for 30 min each, and EdU was labelled with the Click-iT Plus reaction cocktail. The cells were imaged using a confocal microscope with a 20/0.8 NA objective and small voxel size. An area of 3,072 × 3,072 pixels was imaged for each biological replicate.

For differentiation of 3T3-L1 cells to white adipocytes, cells were seeded in 12-well plates at a density of 1 × 105 per millilitre. The medium was refreshed until cells were confluent. Two days following cell confluence, induction medium (10% FBS, 500 μM 3-isobutyl-methylxanthine and 1 μM dexamethasone in DMEM) with or without 1 μg ml−1 insulin was added to each well; 3 days later the induction medium was replaced with maintenance medium (DMEM with 10% FBS with or without 1 μg ml−1 insulin). Maintenance medium was used for 2 days a day. The total time for each well was 8 days. NPY treatments in experimental groups started when the induction medium was added and lasted throughout the whole differentiation process.

The 3T3-L1 preadipocyte cell line was a gift from R. Klemm at the Department of Physiology, Anatomy and Genetics, University of Oxford, and used without further authentication or testing for mycoplasma contamination. The complete medium for cell culture includes high-glucose DMEM with glutamate (ThermoFisher, catalogue no. 41965039) and 10% calf serum (Sigma, catalogue no. 12133 C). For subculture of preadipocytes the medium was transferred, and cells were washed with 1 ml of prewarmed 0.05% trypsin (ThermoFisher, catalogue no. 25300054) and digested with 200 μl of trypsin at 37 °C for 5–10 min. Afterwards, digestion was stopped and cells resuspended using the complete medium.

To perform in vitro coculturing experiments, cells were seeded at 5 × 104 per millilitre on glass coverslips and the indicated concentrations of PDGF-BB (R&B, catalogue no. 220-BB) and NPY (Cayman Chemical, catalogue no. CAY15071) were added to cells 24 h following cell seeding. The cells were collected 5 days later and either injected with 4% PFA or lysed with Trizol.

The first step in preparing a sample for sorting is to remove red blood cells and then treat them with a Fc block. 14-9161-73) before staining with antibodies. Before immunolabelling for intracellular markers, cells were fixed and permeabilized using the eBioscience Intracellular Fixation and Permeabilization Buffer Set (ThermoFisher, catalogue no. 88-8824-00). Cell sorting was performed using a BD FACSAria III sorter, and flow cytometry data were acquired with either a BD FACSAria III sorter or a BD LSRFortessa X20 cytometer with BD FACSDiva v.6.0 software. Cytometry data were analysed using FlowJo v.10.8.1.

For terminal blood collection, mice were euthanized by intraperitoneal injection of 10 μl g−1 pentobarbital and blood was then collected from the left ventricle using a 25 G needle and a syringe coated with 100 mM EDTA. Blood was then placed in tubes with 5 μl of 100 mM A supernatant was moved to fresh tubes with aprotinin and a final concentration of 500,000 IUml, after it was seperated from blood cells. The concentration of NPY in the blood of a mouse was determined using an NPY ELISA kit. EZRMNPY-27K). The data was recorded using a FLUOstar Omega microplate reader.

The extracts from iWAT were described previously. The iWAT was placed in a homogenizing tube with 50 M RIPA lysis buffer. 500,000 IU aprotinin is found in the catalogue no. A6103-1MG) and homogenized using a Precellys 24 homogenizer. Lipid in the tissue homogenize was removed by centrifuging at 20,000× rcf at 4 °C for 15 min, and the clear portion retained. The process was repeated three more times.

Source: Sympathetic neuropeptide Y protects from obesity by sustaining thermogenic fat

Antibody Staining Studies on the Thermogenic and Adipogenic Genes of the Nucleotide Rat, Anti-NLVAP, Rabbit Anti-DE and Anti-EPHB4

The data was collected from the CFX 96 Real-Time PCR Detection System (bio- rad) and the Tuba Green PreMix Ex Taq II.

The ΔCt method was used to quantify gene expression using the following formula: relative expression = 2^ (−(Cttarget gene − Ctreference gene)). The ΔΔCt method was used to compare the thermogenic and adipogenic gene expression shown in Fig. 5b and extended data can be found in the table. 9l,m and 10m are included.

The following primary antibodies were used for immunofluorescent staining: rat anti-CD31 (BioLegend, catalogue no. 102501, MEC13.3, 1:100 dilution), rat anti-PLVAP (BioLegend, catalogue no. 120503, MECA32, 1:100 dilution), rabbit anti-DES (abcam, catalogue no. rabbit anti-NPY, 1:500 dilution, is in the Cell Signaling catalogue. rabbit anti-TH, and chicken anti-TH were both contained in 1:500 dilution. Ab151, 1:500 dilution), mouse anti-NPY1R. sc-393192, 1:200 dilution), rat anti-PDGFRα (BioLegend, catalogue no. 135902, APA5, 1:200 dilution), rabbit anti-TAGLN (abcam, catalogue no. Cy3 anti-SMA is a reagent used for anti-SMA. C6198, 1A4, 1:250 dilution), goat anti-SOX17 (R&D, catalogue no. AF1924, 1:250 dilution), goat anti-EPHB4 (R&D, catalogue no. The rabbit anti-UCP1 is a species that has a dilution of 1:500.

Source: Sympathetic neuropeptide Y protects from obesity by sustaining thermogenic fat

Optical microscopy analysis of human epididymal blood adipose tissue using anti-Stokes Raman scattering and dextran

Animals were anaesthetized with ketamine (100 mg kg−1) and xylazine (10 mg kg−1) and a small incision was made to expose epididymal white adipose tissue for microscopy analysis. The image of the space resolution was obtained using an optical microscope and a confocal L SM 780-NLO. The State University of Campinas has a national Institute of Science and Technology that specializes in cell biology, and all procedures were performed there. Adipocyte images from coherent anti-Stokes Raman scattering were acquired using two lines of lasers in wavelengths λpump = 803 nm and λStokes = 1,040 nm, and fluorescence images were acquired by two-photon excitation fluorescence using the exogenous fluorescence dye tetramethylrhodamine isothiocyanate dextran (Sigma, catalogue no. T1162) The Stokes wavelength is 1,040 nautical miles. The video was started after identification of blood vessels and acquisition of an initial image, and after ten frames, 50 l (30 km2 of treacly) of the drug. T 1154 was inserted into the mouse’s central nervous system. The video continued recording for a while to capture the vessel fluorescent and the leak. Fluorescence intensity over time was analysed for the whole duration of the video. A static image was analysed following dextran administration using Fiji47. The evaluation was done by comparing the region inside the vessel and the one outside the vessel. Fluorescence intensity was measured in both areas, and the intensity ratio between the tissue and within the vessel was calculated. Blood flow was calculated using the average of the raw intensity of dextran inside the artery over 100 s immediately following stabilization of the dextran signal after administration.

Images were acquired using a Zeiss LSM880 confocal microscope with Zen-black software (v.2.1). Samples were imaged using either (1) a ×10/0.45 numerical aperture (NA) objective with a voxel size of 1.04 μm (x), 1.04 μm (y) and 3.64 μm (z), (2) a ×20/0.8 NA objective with voxel size of 0.52 μm (x), 0.52 μm (y) and 1.00 μm (z) or (3) a ×63/1.4 NA oil-immersion objective with a voxel size of 0.13 μm (x), 0.13 μm (y) and 1.00 μm (z). There were solid-state lasers of 405, 571, and , as well as an anaglyph 488 laser that were used.

The whole-mount, PFC-fixed ganglia were dried once in each of 30, 50, 70 and 90 and then again in 100 and 30% Ethanol, with shaking for 20 min at each concentration. Ganglia were then digested with 1.5 U ml−1 dispase-1 (Roche, catalogue no. 04942078001), 0.5 mg ml−1 collagenase (Sigma, catalogue no. C2674) and 300 μg ml−1 hyaluronidase (Sigma, catalogue no. H3884) diluted in PBS, with shaking at 37 °C in a water bath for 30 min. Ethanol-treated ganglia were blocked in blocking solution (3% bovine serum albumin, 2% goat serum, 0.1% Triton X-100 and 0.1% sodium azide in PBS) for 2 h and then incubated with primary antibodies diluted in the permeabilizing buffer for 3 days at 4 °C. ganglia were put into the permeabilizing buffer for 3 more days at 4 C. Finally, ganglia were dehydrated once each in 30, 50, 70 and 90% ethanol and twice in 100% ethanol then cleared using ethyl cinnamate (ECi). There was a slide sandwich filled with ECi.

The samples were processed in multiple batches in 70, 80, and 90% and then in 100% ethanol, xylene and 60 C wax. A sample was attached to wax and mounted on a cover and dried in an oven at 45–50 C. After staining, samples were deparaffinized twice in 100% xylene for 5 min, and then dehydrated again in 100% alcohol, 95 and 80% water, and then again in 95 and 80% booze, for 10 min. Adding 0.05% trypsin to each slide and incubating at 37 C resulted in the antigen retrieval. It was the same procedure for samples to be stained as it was for fixed cells.

The samples were frozen at -20 C in the OCT and embedded in the catalogue. The samples were cut into sections and mounted on charged slides. 631-0108), followed by staining using the same procedure as for fixed cells.

tissue from mice which had been perfused with 20ml of PBS and 4% paraformaldehyde were fixed in at least 4 h. The cells were washed with PBS and fixed with 4% PFC for 4 h. After being stained at 4 C for overnight, the bundles were put into apermeabilizing buffer, where they were first screened with primary and secondary antibodies. The cells were first submerged in the permeabilizing buffer at room temperature for 1h and then stained overnight with primary antibody in the permeabilizing buffer. Samples were then washed and stained with DAPI and secondary antibodies for 1 h at room temperature. Sympathetic fibres or fixed cells were then mounted on slides with Fluoromount-G mounting medium (ThermoFisher, catalogue no. 00-4958-02).

For viral injection, mice were anaesthetized with avertin (240 mg kg−1 intraperitoneally) and fixed on a stereotaxic holder (RWD, Life Science). Either AAV2/9-h Syn-Cre-EGFP-WPRE-pA can be found in the catalogue. S0230-9, 2 × 1,012 viral genomes ml−1, 200 nl per side) or AAV2/9-hSyn-EGFP-WPRE-pA (catalogue no. S0237-9, Taitool, 2 × 1,012 viral genomes ml−1, 200 nl per side) was bilaterally injected into the nucleus tractus solitarius (NTS) (NTS coordinates anteroposterior, mediolateral, dorsoventral −7.5, ±0.35 and −4.75 mm, respectively) of Npyflox/flox mice.

Source: Sympathetic neuropeptide Y protects from obesity by sustaining thermogenic fat

A study of the acclimatization of Npy-hrGFP and ThCre mice with an Optris thermocamera

thermoimaging was used for animals aged 12 to 14 weeks. The data was recorded using an Optris thermocamera with a standard 61 lens and analysed with FOTRIC software. Animals were single housed and placed under the thermocameras, with their back shaved to expose the skin above iBAT. Animals were acclimatized for 4 days in individual cages with ad libitum access to food and water at room temperature under a light cycle and a dark cycle before a 14-h fast. The mice had access to water.

iBAT temperature was recorded every 1 s over a 6-day period by storing the temperature of the warmest pixel in view using the software provided by the camera manufacturer (rel. 2.0.6, Optris PIX Connect). The average temperature for iBAT was 14 and 0 h.

Animals aged 6 weeks were analysed for oxygen consumption, carbon dioxide production, energy expenditure and respiratory exchange rate using an indirect calorimetry system (Panlab, Harvard Apparatus, LE405 Gas Analyzer and Air Supply & Switching). The animals were kept in individual cages under controlled room temperature and 50% humidity after a 12/12/2018 h light/dark cycle with water and food. The data was collected after a 2 day acclimatation period. Animals’ body weight (g) and food (g) were measured before entering and after exiting the cage. The CalR Web-based Analysis tool for Indirect Calorimetry Experiments is used to make graphs and statistical analysis of results.

ThCre mice (B6.Cg-7630403G23RikTg(Th-cre)1Tmd/J, stock no. 008601) and Cx3cr1GFP/+ mice (Cx3cr1tm1Litt/LittJ, stock no. 008451) were purchased from the Jackson Lab. The I. Kalajzic Laboratory at the Department of Reconstructive Science, University of Connecticut agreed to donate mice under the material transfer agreement. Tissues of NPY-GFP mice (B6.FVB-Tg(Npy-hrGFP)1Lowl/J) were sourced from the Tamas Horvath Laboratory at Brandy Memorial Laboratory, Yale University. M. Roberts is the director of the Department of Otolaryngology–Head and Neck Surgery at the University of Michigan. The ThCre mice were crossed with Npyflox mice to create NPY-cKO mice. Diet-induced obesity was achieved by giving mice an HFD. When they were 7 weeks old, this feeding regime was in place for 10 weeks. The body weight of each mouse and food consumption in each cage were recorded weekly. All mice were group housed in standard housing under controlled room temperature (21–23 °C) and 50% humidity under a 12/12 h light/dark cycle and given access to diet and water ad libitum. All experimental procedures were performed on living animals in accordance with the UK ANIMALS ACTS 1986 under the project licence (PPL no. P80EDA9F7) and personal licences granted by the UK Home Office. The University of Oxford had an ethical review panel.

Experimental Reproducibility of scRepli-seq FASTQ Data: Selection, RT Analysis, and Binarization of 1- and 2-cell Embryos

Experimental reproducibility was demonstrated as follows: Fig. 1g, two independent experiments; Fig. Two (2-cell) and three (1-cell, 4-cell) independent experiments were shown in the figure. 2l, two independent experiments; Fig. 3b, two independent experiments; Fig. 3c, four independent experiments; Fig. 3g, two (2-to-4-cell and 16-to-32-cell) and three (4-to-8-cell and 8-to-16-cell) independent experiments; Fig. 3h, two independent experiments; Fig. There are two independent experiments, two independent experiments, and four independent experiments.

In brief, after NGS, the raw scRepli-seq FASTQ files were processed for adapter trimming of both Illumina and SEQXE adapters, mapped to the mm9 reference genome and we filtered out the duplicated reads and reads that overlapped with the mm9 blacklists as described previously16. In order to control the quality of scRepli-seq data, we used a screening method called MAD-score-based screening. More than 90% of cells in each sample passed these criteria. To generate log2[median] single-cell RT profiles, we counted the reads in sliding windows of 200 kb at 40 kb intervals after normalizing S-phase data with AneuFinder’s correctMappability command based on G1 control without karyotype defects in each strain. The binarization using 80 kb or 400 kb (haplotype-resolved analysis) windows was performed using the findCNVs command in AneuFinder as described previously16. For 4-cell embryos, we applied the 1-somy mode for early-S-phase cells and the 2-somy mode for mid/late-S-phase cells. The 2-somy mode was used for the binarization of 1- and 2-cell embryos. As such, the overall binary profile will become blue (replicated) if there is no copy-number variation; likewise, if we use the ‘1-somy’ mode for the analysis of 1- and 2-cell embryos, it will be all yellow (unreplicated) instead of blue (Extended Data Fig. 2c . It would be confusing if we used a third colour to describe the peculiar replication regulation of 1- and 2-cell S phase. Thus, to highlight the binarization failure and the unconventional replication regulation in 1- and 2-cell-stage embryos, we decided to use the ‘2-somy’ mode and blue (replicated) to describe the copy-number state of the majority of bins (Figs. There are 1d and 2f figs. 1g, 2e,h and 4a). The percentage replication scores were calculated using the data from binarized scRepli-seq, which excludes chromosomes X. Averaged scRepli-seq profiles shown in Fig. There is 1b and extended data. The cells that produced 1d were those with 70% or more of the replication score. To identify the heterogeneously late-replicating domains. 2f and Extended Data Figs. scRepli-seq profiles were obtained from the S phase and used to calculate the average profile. The tag-density profile was created using AneuFinder in sliding windows at 40 and 200 kb intervals. t-S NE was used to analyze the scRepli-seq data. For the RtSNE, the log2[median] score of the mid-S cells was used to derive the replication scores.

The first component of the Hi-C data was computed using the published mapped Hi-C datasets of sperm68 and cumulus60 cells. The genomic coordinates of PC1 profiles were converted from mm10 to mm9 using the liftover tool (UCSC Genome Browser). The A/B compartment categories are those containing 25% of the PC1 values, from the strongest A to the weakest B.

After linearization of the template plasmid, mRNA was synthesized using the mMESSAGE mMACHINE KIT (Ambion). Synthesized RNAs were stored at −80 °C until use. In vitro-transcribed mRNAs (0.9 pl of 150 ng µl−1 mEGFP-SLX4, 0.9 pl of 150 ng µl−1 mEGFP-PCNA, 0.9 pl of 150 ng µl−1 Major-satellite-mClover and 0.9 pl of 35 ng µl−1 H2B-mCherry) were microinjected into 1-cell embryos. Live-cell image was performed with some modifications. In this example, the Carl Zeiss 40 C- Apochromat 1.2NA water-immersion objective lens was controlling the confocal microscope. For major-satellite imaging, 17 confocal z sections (every 1.5 µm) of 512 × 512 pixel xy images covering a total volume of 30.30 × 30.30 × 24.00 µm were acquired at 2 min 15 s intervals for at least 3 h just after nuclear envelope breakdown. 17 confocal sections of 512 512 xy images covered a total volume of 30.40 30.30 32.00 m were obtained at 3 min intervals for at least 10 h. Every 3 m, 29 confocal z sections were obtained from the 1-cell to 16-cell images, covering a volume of 84.85 84.85 84.00 m. In the pictures. There is an extended data fig. 7a, to achieve high-resolution live imaging while minimizing phototoxicity, we selected and imaged blastomeres (cells) that were just entering M phase and close to the objective lens, up to two blastomeres (cells) per embryo.

For the experiments described in Extended Data Fig. 5d–i, MC12 cells were cultured on a glass-bottomed dish, treated with 0.5 μM nocodazole for 17 h to synchronize in prometaphase and subjected to 3 μg ml−1 aphidicolin treatment for 3 h. After a few days at the G1 and S-phase borders, cells were cultured again with or without 10mM 2-aminopurine. The cells were stained with EdU for 60 minutes and labeled with 20 M. after the 2-to-4-cell or 4-to-8-cell division (that is, in early S phase) were treated with 3 μg ml−1 aphidicolin with or without 2-AP for 5.5 h followed by reagent removal and EdU staining.

Source: Embryonic genome instability upon DNA replication timing program emergence

Measuring IOD between mobile fork class (iammobile) forks of Alexa Fluor 647 DNA fibres

Images of DNA fibres were subjected to auto-thresholding with the ‘minimum’ or ‘default’ algorithm in Fiji software. Individual DNA fibres were identified as a series of linearly arranged Alexa Fluor 647 (ssDNA) ‘dot’ signals (Fig. 2h). The thresholded fibres containing both alexa 488 and 594 were categorized by their class forks. The immobile class forks were defined as those with single dot signals of IdU + CldU or CldU with gaps between dots, reflecting extremely slow fork movement. The gaps were defined as those that had at least one dot of the ssDNA signal. I am mobile. The mobile class forks were defined as those with a series of dot signals (≥2 dots) of IdU + CldU that contain a series of dot signals (≥2 dots) of CldU on either side of the same DNA fibre (Fig. 2h (mobile)). The intermediate class forks were defined as those with an intermediate character between the two other classes; these fibres also contained dot signals of IdU + CldU and CldU with gaps between dots but also contained some IdU + CldU single colour signals (that is, IdU-only regions) in between these dot signals. The IOD measurement method on the mobile fork class fibres is provided in Extended Data Fig. 5b. The IOD between the immobile forks was calculated by measuring the distance between the brightest pixels in the centre of the dots using the Fiji software. When determining the fork speed of the mobile forks, the tracks flanked by the IdU tracks were identified, their lengths were measured, and divided by the second pulse. The tracks that did not have ssDNA signals before them were not included in the fork speed measurement. For the immobile fork speed measurement, the details are provided in Supplementary Note 2.

The mean signal intensity for H3K9me2, p-CHK1 and H2AX was obtained from the levels of histone H3. We then subtracted the mean cytoplasmic signal intensity (Ime_cyto), which was obtained from a region near the nuclei, from the Ime_nuc value (Ime_nuc − Ime_cyto). Similarly, we determined the histone H3 level within the same nuclei (IH3_nuc − IH3_cyto). Finally, we calculated the ratio between the two values (Ime_nuc − Ime_cyto)/(IH3_nuc − IH3_cyto).

Embryos were fixed with 2% paraformaldehyde in PBS-polyvinyl alcohol (PVA) (pH 7.4) for 30 min. After blocking and permeabilization in PBS-PVA containing 1 mg ml−1 BSA (PBS-PVA-BSA) and 0.1% Triton X-100, the embryos were incubated with appropriate primary antibodies overnight at 4 °C, washed several times in PBS-PVA-BSA and incubated with secondary antibodies for 90 min at room temperature. 40 g of Hoechst 33343 was counterstained with the same genetic material. Finally, the embryos were washed and transferred to BSA-PVA for imaging with a Zeiss LSM780 confocal microscope. The mouse anti-H2A.X was one of the primary antibodies used. The secondary antibodies were Alexa Fluor 488 goat anti-mouse IgG (H+L) (A11029); goat anti-rabbit IgG (H+L) (A11034); Alexa Fluor 555 goat anti-mouse IgG (H+L) (A21424) (1:400, Invitrogen).

scRepli-seq experiments using mouse embryos were performed as previously reported with slight modifications. In brief, unfixed single blastomeres (derived from BDF1 or B6MSM strain embryos) were collected into 8-well PCR tubes with 6 μl cell lysis buffer (288 μl of H2O, 2 μl of 10 mg ml−1 proteinase K (Sigma-Aldrich, P4850), 32 μl of 10× single-cell lysis and fragmentation buffer (Sigma-Aldrich, L1043)), incubated at 55 °C for 1 h and then at 99 °C for 4 min for gDNA isolation and fragmentation. For scRepli-seq analysis, we analysed all blastomeres of an embryo unless there was accidental damage to the cell/sample. The scRepli-seq experiments were after EdU staining. 3b,c), after taking the photographs of EdU-stained cells (according to the protocol in Extended Data Fig. 3a), the cells were manually collected by a mouth pipette under the microscope into 12 μl of cell lysis buffer and incubated at 55 °C for 16 h (not 1 h). The remaining whole-genome amplification process (SeqPlex enhanced DNA amplification kit, Sigma-Aldrich, SEQXE) and next-generation sequencing (NGS) library construction (NGS LTP library preparation kit, KAPA, KK8232) were basically performed according to the manufacturer’s instructions. The samples were processed for Next Generation Sequencing on the Illumina Hiseq 1500 or Hiseq X Ten system.

The temperature, humidity and light cycle of mouse cages were maintained at 20–24 °C, 45–65% and 12 h–12 h dark–light, respectively. Mature oocytes were collected from the oviducts of eight- to ten-week-old female mice that had been induced to superovulate with 5 IU of equine chorionic gonadotropin (eCG, ASKA Pharmaceutical) followed by 5 IU of human chorionic gonadotropin (hCG; ASKA Pharmaceutical) 48 h later. Cumulus-oocyte complexes were collected from the oviducts approximately 16 h after hCG injection. Cumulus-oocyte complexes were placed in M2 medium and treated with 0.1% (w/v) bovine testicular hyaluronidase. After several minutes, the oocytes were washed twice and moved to a variety of Mediums. Mature metaphase II (MII) oocytes were subjected to in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) or SCNT. Delayed or arrested embryo’s were not included in further analysis. In vivo developed zygotes and embryos were collected from the oviducts of pregnant mice that had been induced to superovulate and mated with male mice. For in vitro development, in vivo developed zygotes were cultured in CZB. The dataset is not suitable for the assessment of variability between males and females. When indicated, the embryo were cultured in the presence of the specified nucleosides or at a final concentration of 2. The extended data fig. embryos were monitored under the microscope every 30 min and transferred to the medium containing aphidicolin if they were found to be in G1 after completing cell division. Liveimaging was used to observe the M phase. 7a. Embryos that had just completed the 4-to-8-cell division were collected for scRepli-seq as in Extended Data Fig. 7b.

All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Committee of Laboratory Animal Experimentation. B6D2F1 (C57BL/6 × DBA/2) and C57BL/6 mice, aged 8–10 weeks, were used to produce oocytes and sperm. For allele-specific analysis, C57BL/6 female mice and MSM/Ms male mice were used to produce oocytes and sperm, respectively. To eliminate the effect of individual differences between mice, multiple mice were used in each experiment as follows. Figure 1b, 2 (4-cell), 4 (8-cell), 2 (16-cell) and 4 (ICM and TE) mice; Fig. There are 10 mice per cell, 2-cell and 4-cell. Fig. 2d, 4 (1-cell), 3 (2-cell) and 3 (4-cell) mice; Fig. 2f, 3 (1-cell), 3 (2-cell) and 4 (4-cell and 8-cell) mice; Fig. 3b, 4 in vitro and 2 in vivo (2-to-4-cell), 8 in vitro and 7 in vivo (4-to-8-cell), and 4 in vitro and 2 in vivo (8-to-16-cell) mice; Fig. 15 mice each (2-to-4)-cell, 4-to-8-cell, 8-to-16-cell and 16-to-32-cell); 6 (2-to-4-cell aphidicolin 30 ng ml1, and 60 ng ml1), and 8 (2-to-4-cell aphidICOlin 75 ng ml1) are included in 7a.