Single cell RNA Seq profiling of human pre …...Single-cell RNA-Seq profiling of human...

Single-cell RNA-Seq profiling of human pre-implantation embryos and

embryonic stem cells

Liying Yan1,2,5, Mingyu Yang1,5, Hongshan Guo1, Lu Yang1, Jun Wu1, Rong Li1,2, Ping

Liu1, Ying Lian1, Xiaoying Zheng1, Jie Yan1, Jin Huang1, Ming Li 1, Xinglong Wu1,

Lu Wen1, Kaiqin Lao4, Ruiqiang Li1,3*, Jie Qiao1,2*, Fuchou Tang1*

1Biodynamic Optical Imaging Center & Center for Reproductive Medicine, College

of Life Sciences, Third Hospital, Peking University, Beijing 100871, P. R. China 2Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, P.

R. China 3Genetic Systems, Applied Biosystems, Life Technologies, Foster City, California,

USA 4Peking-Tsinghua Center for Life Sciences, College of Life Sciences, Peking

University, Beijing 100871, P. R. China 5These authors contributed equally to this work. *Correspondence should be addressed to: R. L. ([email protected]), J. Q.

([email protected]), or F. T. ([email protected])

Nature Structural & Molecular Biology: doi:10.1038/nsmb.2660

Supplemental Materials

zygote 2- 4-cell 8-cell Blastocyst

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Supplementary Fig. 1. Expression dynamics of known RefSeq genes during human

pre-implantation development. (a) Number of RefSeq genes showing up- or

downregulation during pre-implantation development (fold change, FC>2 or <0.5, p-

value <0.01). (b) Heatmap of the genes showing differential expression between 4-

and 8-cell stage embryos. (FC>4 or <0.25, p<0.01) (c) Correlation plots for the

expression of RefSeq genes for individual blastomeres from three 2-cell-stage

embryos. The correlation coefficient between blastomeres within the same 2-cell

embryo is higher than 0.99, indicating that the technical variation is reasonably low.

(d) Correlation plots for the expression of RefSeq genes of single mature oocytes and

single blastomeres of pre-implantation embryos.


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Supplementary Fig. 2. Global expression profile of known and novel lncRNAs

during human pre-implantation development. (a) Correlation plots of the expression

of known lncRNA genes for individual blastomeres from three 2-cell-stage embryos.

(b) Correlation plots of the expression of known lncRNA genes for mature oocytes

and blastomeres of pre-implantation embryos. (c) Correlation plots of the expression

of novel lncRNA transcripts for individual blastomeres from three 2-cell-tage

embryos. (d) Number of novel lncRNA transcripts showing up- or downregulation

during pre-implantation development (Fold Change, FC>2 or <0.5, p<0.01). (e)

Correlation plots of the expression of novel lncRNA transcripts for mature oocytes

and blastomeres from pre-implantation embryos.


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Supplementary Fig. 3. Heatmap and expression pattern of known RefSeq genes

during pre-implantation development. (a) Heatmap of the genes showing differential

expression between the epiblast and trophectoderm lineages of cells in late blastocysts.

(b) Heatmap of the genes showing differential expression between the epiblast and

primitive endoderm lineages of cells in late blastocysts. (c) Heatmap of the genes

showing differential expression between the primitive endoderm and trophectoderm

lineages of cells in late blastocysts. (d) Expression pattern of lineage specific marker

genes in individual cells of blastocysts. EPI: epiblast; PE: primitive endoderm; TE:

trophectoderm.


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Supplementary Fig. 4. Expression of marker genes in individual cells during human

pre-implantation development and derivation of hESCs. (a) Expression of marker

genes (including transcription factors) in individual cells during human pre-

implantation development and derivation of hESCs. (b) Expression of epigenetic

regulator genes in individual cells during human pre-implantation development and

derivation of hESCs. The housekeeping gene RPS6 was used as a control. Each

vertical bar represents the expression (RPKM) of the gene in an individual cell. Each

column represents the expression of different genes within the same cell. TE:

trophectoderm; PE: primitive endoderm; EPI: epiblast; P0: hESCs of passage #0; P10:

hESCs of passage #10.


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Supplementary Fig. 5. Gene expression dynamics of blastocysts and hESCs. (a)

Expression pattern of pluripotency-related genes in human embryonic stem cells

(hESCs) and human late blastocysts. The RPKM values of the genes are labeled at the

top of the highest bar for every gene. (b) Unsupervised hierarchical clustering analysis

for single cells of EPI and hESCs. (c) Expression pattern of pluripotency-related

genes in human embryonic stem cells (hESCs) and mouse embryonic stem cells

(mESCs). The RPKM values of the genes are labeled at the top of the highest bar for

every gene.


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Supplementary Fig. 6. Compare between Smart-Seq and our single cell RNA-Seq

methods. (a) Number of genes detected in individual hESCs. (b) Mean read coverage

over transcripts for single cell RNA-Seq data of our technique (n=8) or Smart-Seq

technique (n=8) for human embryonic stem cells. For comparison, we included data

from standard RNA-Seq (n=2) on bulk amount of human embryonic stem cells (non-

amplified)1. Error bar: s.d.


Supplementary Fig. 7 Characterization of the hESC cell line and the lineage

differentiation of the blastocysts. (a) Unsupervised hierarchical clustering analysis for

single cells at morula and blastocyst stages. (b) Principal component analysis (PCA)

of the transcriptomes of single cells at morula and blastocyst stages. (c) The karyotype

(46, XY) of the hESC line used in the present study and Western blot of the

pluripotency markers OCT4, NANOG, and SOX2 in the analyzed hESCs. HeLa cells

were used as a control. (d) Immunostaining for TRA-1-60 (green) and corresponding

nuclear staining (PI staining; red) in hESCs. (e) Teratomas formed by the hESC line

analyzed in SCID mice. Ectoderm tissues (chromatophores, neuroepithelial) in

teratoma sections. Mesoderm tissues (bone) in teratoma sections. Endoderm tissues

(pseudostratified columnar epithelium) in teratoma sections. Scale bar: 20 µm.


Supplementary Note

As one example, the seven transcript isoforms of RING-H2 finger protein ANAPC11,

produce two different proteins with 84 and 196 amino acids in the same hESC,

respectively. The expression level of the mRNAs for these two proteins is RPKM 181

and 16, respectively. It is known that ANAPC11 is a core component of anaphase-

promoting complex and together with E2 enzyme UBC4, it is sufficient to

ubiquitinate a wide variety of substrates during cell cycle regulation2.

We found that the DNA methyltransferases DNMT3A and DNMT3B and the histone

deacetylase HDAC7 are strongly upregulated from epiblast cells to passage #0 hESCs

(Supplementary Fig. 4b. Conversely, DNMT3L is highly expressed in epiblast cells

but is downregulated by over a hundred fold in hESCs, which implies that DNA

methylation-related factors is likely involved in the derivation of hESC cells from

epiblast cells (Supplementary Fig. 4b). In fact, of the 113 known epigenetic

regulators and chromatin remodeling factors, 21 show clear changes from epiblast

cells to passage #0 hESCs (Supplementary Table 8). Therefore, our results show that

during the derivation process of hESCs, the most dramatic changes in the expression

of epigenetic regulators occur at the earliest time point during the first ten days in

culture, which can probably be attributed to the accommodation of the cells to the

culture conditions, such as the addition of serum and cytokines.


References 1. Gifford, Casey A. et al. Transcriptional and Epigenetic Dynamics during

Specification of Human Embryonic Stem Cells. Cell 153, 1149-1163 (2013). 2. Gmachl, M., Gieffers, C., Podtelejnikov, A.V., Mann, M. & Peters, J.-M. The

RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphase-promoting complex. Proceedings of the National Academy of Sciences 97, 8973-8978 (2000).


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