Supporting Information Appendix for · Supporting Information Appendix for ... Global differential gene expression analysis by mRNA-seq. ... Color chart representation of 20bp of

1 | P a g e S u p p o r t i n g I n f o r m a t i o n

Supporting Information Appendix for

“Charting Brachyury-mediated Developmental Pathways During Early Mouse

Embryogenesis”

Table of Contents:

Supplementary Figure 1 - 11.

Supplemental Methods.

Supplementary References.


SUPPLEMENTARY FIGURES


Fig. S1. In vitro Primitive Streak Induction from ES cells

(A) Scheme for ES cell Differentiation: 2 days after culturing in serum-free medium,

embryoid bodies were subjected to a 2 day treatment with Activin A.

(B) Western blots examining Brachyury protein levels after differentiation. ES cells and

EBs without Activin A treatment served as controls

(C) Global differential gene expression analysis by mRNA-seq. The log10-scale FPKM

values of gene for both ES cells and differentiated EBs are plotted. Dark green, 10 fold

down; Light green, 100 fold down; Orange, 10 fold up; Red, 100 fold up relative to the

expression levels in ES cells. See also Dataset S1.

(D) Immunofluorescent staining to analyze primitive streak gene induction (Foxa2, Sox17

and Brachyury) at EB day 4.

(E) Gene-centered Annotation analysis of Brachyury ChIP-seq peaks by CEAS (1)

(F) Brachyury ChIP-seq data set associated gene feature enrichment compared to

random genomic background by CEAS (1)


Fig. S2. Gene Induction During in vitro Primitive Streak Formation

(A) qRT-PCR analysis of checking the induction of primitive streak genes (Gsc and

Brachyury) when different concentrations of Activin A were used.

(B) Selected differentially expressed genes in our mRNA-seq data (Fig. S1C) were

validated by qRT-PCR.


Fig. S3. Mapping Ultra-fine Brachyury-binding Events in the Genome by ChIP-exo

(A) Tracks comparing the length of a ChIP-seq region with that of a ChIP-exo peak.

(B) Histogram showing the distribution of lengths of ChIP-exo protected regions (light-red)

and compared to the lengths of ChIP-seq regions (cyan).

(C) Top, Average raw sequencing reads per base pair across 100bp regions around Motif

1. Bottom, Raw sequencing reads (Forwards, blue; Reverse, green) around genomic


regions (1K dataset, See Supplemental Methods) containing the “TCACA” DNA motif (M1)

which matches the first 5 bases of the canonical Brachyury-binding sequence.

(D) Color chart representation of 20bp of sequence located around the midpoint of Motif

1 ordered as in (C).

(E) Multiple Brachyury-binding events were observed at the proximal enhancer (PE)

regions of Foxa2, Cer1 and Wnt8b. The geometric relationship between the PE and the

TSS of the gene is presented at the up-right corner of each ChIP-seq track.


Fig. S4. Genomic and Biochemical Analyses of ChIP-exo Peaks

(A) Probe design for the EMSA assay: C7bp, 7bp of the Brachyury consensus sequence;

F5bp, the first 5 base pairs of C7bp.

(B) EMSA assays to assess the binding affinity of Brachyury to C7bp and F5bp probes.

(C) Super-shift were performed with either IgG (lane 2 & 4) or anti-Brachyury antibody

(lane 3 & 5)


Fig. S5. Multiple DNA Motifs were Enriched Around Exo-peaks

(A) Left, Raw sequencing reads (Forwards, blue; Reverse, green) of 200bp genomic

regions containing DNA motif 5 that were de novo recovered by gimme-motif using the

1k datasets, centered by the motif midpoint. Right, Color chart representation of 20bp of

sequence located around the midpoint of each motif ordered as in the left.

(B) Number of 50bp genomic regions surrounding exo-peaks (11k dataset) that contain

three motifs.


Fig. S6. Brachyury as a Transcription Activator

(A) Differential gene expression analysis with mRNA-seq to identify up/down regulated

genes upon Brachyury depletion. We only considered genes that were significantly

changed in both shRNA 1 and shRNA 2 treated samples. The Log2 scale values of FPKM

for genes in both control and shRNA samples were plotted in the graph. Dark green, 2

fold down; Light green, 8 fold down; Orange, 2 fold up; Red, 8 fold up.

(B) Seq-region and differential gene expression association: ChIP-seq regions that are

within 5kb of TSS’s were assigned to genes genome-wide. 100 genes were randomly

sampled from up-regulated (light red), down-regulated (green) or unchanged genes (blue).

Histogram distributions of the number of seq-regions associated with each category are

plotted.

(C) Dual luciferase reporter assay to analyze the enhancer activities of the 12 Brachyury-

bound regions in the presence of either Brachyury or RFP. 8 out of the 12 regions showed

significant enhancer activities in synergy with Brachyury.

(D) Brachyury K/D EB differential gene expression data (Dataset S1) are correlated with

Brachyury-target gene information (Dataset S3) to identify functional in vivo Brachyury


targets. 63 up- and 52 down- regulated Brachyury target genes were identified (listed in

Dataset S4). 2 fold up/down regulated genes are considered in this analysis.


Fig. S7. Brachyury Functions as a Transcriptional Activator

(A) 12 Brachyury-bound seq-regions (For genomic locations, see Dataset S5) were

cloned into pGL-4.23 [Luc2/minP] vector and were fully sequenced. The restriction

digested fragments were resolved on the Gel. DNA marker, 1 Kb Plus DNA Ladder

(Invitrogen)

(B) Likewise, 4 repeats of Brachyury-binding site were able to elevate luciferase reporter

expression in the presence of Brachyury.


Fig. S8. Brachyury ChIP-seq Tracks at Key Developmental Genes

(A-D) Brachyury ChIP-seq exemplary tracks at key developmental genes (Mab21l2,

2.703kb; Rb1, 130.290kb; Sox17, 2.808kb; Aplnr, 3.564kb; Amotl2, 16.616kb; Fgf4,


3.858kb; Wnt3a, 42.71kb). Y axis limit is from 3 to 50 reads for chip-seq data. Auto-scale

is used for mRNA-seq.

Note: Brachyury is highly conserved among all bilaterian animals. Several developmental

genes have been identified as Brachyury direct targets in different model organisms. For

example, Fgf4 is a bone fide target of Brachyury in Xenopus (2). Cyp26a1 is a Brachyury

target in Zebrafish (3). Although Fgf4 and Cyp26a1 are still moderately expressed after

differentiation, no Brachyury sites were found around these two genes (C-D), suggesting

that these regulations are probably evolutionarily divergent. Interestingly, consistent with

the previous report (4), we also found that Wnt3 was down-regulated by Brachyury K/D.

However, no Brachyury sites were located in proximity of the gene (D), implying an

indirect regulation.



Fig. S9, Extension data for Fig. 3

(A) Immunofluorescent staining to analyze primitive streak gene induction (Foxa2, Sox17

and Brachyury) at EB day 4 without or with Brachyury K/D. Control was the same as in +

Activin A lane shown in Fig. S1D. Statistics was listed in (B).

(B) Counting statistics of Brachyury, Sox17 and Foxa2 positive EBs for control and

shRNA A treated samples

(C) Image showing an EB that had separated Brachyury and Sox17 expressing poles.

(D) Positive correlation between the protein levels of Sox17 and Foxa2 (middle

expression levels) when segmented cell areas were determined by the Brachyury channel.

Middle-to-low expression levels of Foxa2 and Sox17 were detected.

(E) Left, In Situ Hybridization (ISH) assay mapping Brachyury, Foxa2 and Sox17 gene

expression in E6.5 and E7.75 Wt embryos. The publicly shared data was obtained from

EMAGE gene expression database under the citation agreement (id; EMAGE: 111, 3674,

587, 104, 255 and 598)

(http://www.emouseatlas.org/emage/) (5).

Right, the spatial gene expression patterns for images in left panel were extracted with

color information by Matlab and tested for overlaps. The expression of Brachyury and that

of Foxa2 overlap at definitive endoderm and node region. The expression of Sox17 and

that of Foxa2 overlap at the definitive endoderm region.



Fig. S10. Brachyury and Foxa2 Embryo Co-Immuno-Staining

(A) Whole mount Brachyury and Sox17 Immuno-staining confocal analysis of E7.5

embryos. Cross section covering primitive streak was selected to show. For other regions,

see Movie S2. Upper 10X magnification (scale bar 100 µm), lower 40X oil magnification

(scale bar 25 µm) of the posterior primitive streak region indicated by the yellow dashed

box in upper panel.

(B) Whole mount Brachyury and Foxa2 Immuno-staining confocal analysis of E7.0

embryos. Cross section covering primitive streak and definitive endoderm is selected to

show. For other regions, see Movie S2. Upper 10X magnification (scale bar 100 µm),

lower 40X oil magnification (scale bar 25 µm) of the node and DE region indicated by the

yellow dashed box in upper panel

(C) Whole mount Brachyury and Foxa2 Immuno-staining confocal analysis of E7.5

embryos. Node/Definitive endoderm region was selected to show. For other regions, see

Movie S2-3. Upper 10X magnification (scale bar 100 µm), lower 40X oil magnification of

DE region indicated by the yellow dashed box in upper panel (scale bar 25 µm). The

anatomy terms of early mouse embryo are reviewed in (6)


Fig. S11. 4-way Differential Gene Expression Test to Characterize Developmental

Deficiencies in Tc/Tc Mouse Embryos

(A) At E8.0, Tc/Tc mouse embryo (Right) was severely shortened compared to the wild-

type embryo (Left)

(B) Non-closure of neural folds and lack of limb-bub were observed in Tc/Tc mouse

embryo (Left) at E10.0


(C) Middle: Diagram of the 4-way comparison for embryos of the indicated genotype and

stage. Wt - Wild-type; Mt – Tc/Tc. Arrow starts with control sample and points at the

treated sample. Total of 4 comparisons were performed. The Log2 scale values of FPKM

for genes in both control and Tc/Tc mutant samples are plotted in the graph. Dark green,

2 fold down; Light green, 8 fold down; Orange, 2 fold up; Red, 8 fold up. The diagram for

the Tc/Tc and WT comparison is presented in Fig. 4D. Selected significantly up and down-

regulated genes are displayed in each graph. All differential gene expression and gene

ontology association data are listed in Dataset S1.

Note:

To fully understand the consequences of Brachyury Loss-of-function, we

conducted a 4-way differential gene expression analysis (C). Specifically, the test

between wild-type samples (Comparison 1, C1) would provide us the baseline information

for normal developmental progress made from E7.5~8.0 to E10.0-10.5. Developmental

deficiencies in the Tc/Tc embryos could then be discerned from the C2 and C3 tests.

Notably, from the C1 comparison, we detected dramatic up-regulation of genes involved

in neural differentiation (Zic1, Foxp2, Neurod4, Olig2 and Neurog1), skeletal muscle

formation (Myog), haematopoiesis (Hbb-y and Hba-x), anatomical structure

morphogenesis (Hoxa6, Hoxc10) and X-chromosome inactivation (Xist). By contrast,

genes involved in gastrulation (T, Cdx2), visceral endoderm (Sox17, Cer1 and Ape),

cardiovascular system development (Foxh1) and pluripotent stem cell maintenance

(Pou5f1) were significantly down-regulated. These differential gene expression changes


correlated well with the rapid axial mesoderm development, somite formation, and

neurogenesis that occur between E8.0 and E10.0.

From the C2 comparison, we found that haematopoiesis (Hba-x), cardiovascular

system development (Gata5, Mef2c, Nkx2-5), limb bud formation (Tbx4), gastrulation

(Fgf8) and notochord development (Shh) were all severely compromised in the Tc/Tc

embryos at E7.5~8.0 (C). In line with these observations, at E10.0~10.5, the fetal blood

(Hba-x, Hba-y), neural differentiation (Zic1, Foxp2 and Olig2, Neurog1 and Neurog4) and

skeleton muscle markers (Myog) were significantly down-regulated. It is also worth noting

that the X-chromosome inactivation effector, Xist, was dramatically up-regulated in the

E10.0~10.5 Tc/Tc embryos as seen in wild type E10.0~10.5embryos, indicating a fair

comparison between these two conditions. However, in contrast to E7.5~8.0,

cardiovasulcar system development in the E10.0~10.5.Tc/Tc embryos appears to be

normal. Next, we found that genes involved in cardiovasulcar system development (Mef2c

and Nkx2-5) were significantly up-regulated in E10.0~10.5 Tc/Tc embryos (Comparison

4, C4). Together, these data suggest that Brachyury-mediated regulation promotes

cardiovascular system induction but may not be necessary for its early development. It’s

interesting to note that cardiovascular lineages originate from lateral plate mesoderm(7).

On the other hand, Brachyury is clearly essential for axial mesoderm related

developmental processes, such as notochord development and neural tube patterning

(8). These results suggest that Brachyury might provide distinct functions for axial and

lateral plate mesoderm development. Since notochord is critical for proper neural tube

induction and formation, the lack of notochord formation and sonic hedgehog signaling

could at least partially explain some of the neural differentiation defects observed in the


Tc/Tc mice. Interestingly however, unlike the in vitro differentiation system, we didn’t

observe significant changes of Foxa2 and Sox17 gene expression in Tc/Tc mouse

embryos (Dataset S1).

We note that Brachyury functional targets detected in vivo versus in vitro only

partially overlap (Dataset S4). This result may be in part due to the fact that In vitro

primitive streak formation is an isolated system with relatively high purity of Brachyury

expressing cells. Under such conditions, it is likely easier to detect Brachyury regulation

of genes whose expression is not specific to primitive streak such as Foxa2, Sox17,

Dusp6 as well as house-keeping genes (Rb1, Rps3a, Rpl22). We suspect that in vivo, the

impact of Brachyury loss-of-function on these genes may be masked by redundant gene

expression from other tissue types in the embryo. On the other hand, in an isolated in

vitro differentiation system, the in vivo developmental and signaling cues from other cell-

types are missing. Thus, it would be less likely for us to detect expression changes of

genes regulated by Brachyury in synergy with external signals. The opposite situation

occurs in vivo where it may be easier to identify expression changes of genes regulated

by Brachyury in conjunction with external signaling pathways. This situation may also

explain why most Brachyury target genes identified in Tc/Tc embryos are related to a

specific signaling pathway and the induction of a specific cell-type (Fgf8, Wnt5b, Msgn1,

Mllt3). One advantage of our study is that the combination of both in vitro and in vivo

approaches might provide a more comprehensive set of potential functional Brachyury

target genes. Taken in aggregate, these studies provide a useful guide for future genetic

studies to achieve a better understanding of the finely balanced cell-lineage commitment

and morphogenic events mediated by Brachyury. Our studies also suggest that the in


vitro differentiation of ES cells could provide a useful system to isolate and study critical

regulatory factors of cell-fate determination and that could be extended to study

transcriptional control mechanisms of other early developmental regulators.


SUPPLEMENTAL METHODS

ES Cell Culture and Differentiation

Mouse D3 (ATCC) ES cells were cultured on 0.1% gelatin coated plates in the absence

of feeder cells. The ES cell medium was prepared by supplementing knockout DMEM

(Invitrogen) with 15% FBS, 1mM glutamax, 0.1mM nonessential amino acids, 1mM

sodium pyruvate, 0.1mM 2-mercaptoethanol and 1000 units of LIF (Millipore). Serum free

in vitro differentiation of EBs was performed as previously described (9) with minor

modifications. Specifically, cells were plated at a concentration of 75,000cells/ml. After 2

days, 20ng/ml Activin A (R&D) was added into medium without dissociation/re-

aggregation. After 2 additional days, EBs were harvested for experiments. As for

electroporation experiment with EB day 2 cells, EBs were first dissociated by trypsin into

single cells and then, we used the Nucleofector Kits for Mouse Embryonic Stem Cells

(Lonza) for electroporation.

Brachyury Antibody Production

For antibody used in Brachyury western blot, staining and ChIP experiments, rabbits were

immunized with Brachyury residues 249-436 GST fusion proteins. It is important to note

that the sequence of the antigen region is specific to Brachyury but not to other T-box

proteins. The antisera obtained were further affinity-purified using MBP-antigen fusion

protein immobilized on Affigel 10/15 resin (Bio-Rad).

Lentivirus-mediated shRNA Knockdown


Anti-Brachyury shRNA A (Sense, 5-CCTTCCTTGATGCCAAAGAAA-3) and anti-

Brachyury shRNA B (Sense, 5-GCCTGTGTCTTTCAGCAAAGT-3) were cloned into

pLKO.1 - TRC Cloning Vector (10). Lentiviral particles were generated by transfecting

TLA-HEK293T cells (Open Biosystems) with a PLKO.1 shRNA vector and packaging

vectors, psPAX2 and pMD2.G (Addgene), according to the RNAi consurtium protocol (10).

Viruses were concentrated using Fast-Trap Lentivirus Purification and Concentration Kit

(Millipore). The titer of virus stock was determined using QuickTiter Lentivirus Titer Kit

(Cell Biolabs. Inc).

Western Blot

Whole cell extracts from ES Cells and EBs were isolated using RIPA buffer that contained

Complete Protease Inhibitor Cocktail (Roche). Protein concentrations were measured

using Bio-Rad Protein Assay against BSA standards. Protein from each sample was

resolved by SDS-PAGE. Primary antibodies used: Brachyury (Custom-made), Sox17

(R&D systems AF1924), Foxa2 (Cell Signaling 8186), beta-tubulin (E7) and beta-actin

(A2228, Sigma). HRP conjugated secondary antibodies (Pierce) were used at a dilution

of 1:5000. Western Lightning Plus–ECL (Perkin) was used for chemiluminescent

detection.

qRT-PCR

Total RNA was extracted with Trizol LS Reagent (Invitrogen), and reverse-transcribed by

SuperScript III First-Strand Synthesis System with oligo-dT primer (Invitrogen). cDNA

http://www.rndsystems.com/pdf/af1924.pdf


corresponding to 10ng of total RNA was used in each iQ Sybr Green Supermix (Bio-Rad)

reaction. Reactions were performed in triplicates on a DNA Engine Opticon 2 Real Time

cycler. Cycle threshold values (log2 scale) were obtained in MJ opticon analysis software

(Bio-Rad). Housekeeping genes, beta-actin and Gapdh, served as internal controls.

Primer information is in Dataset S5.

Immunofluorescence Staining

Whole mount staining was performed for embryoid bodies and embryos in suspension.

EB or Embryo samples were first fixed with 4% paraformaldehyde, permeabilized with

PBST (PBS plus 0.2% Triton X-100) and blocked with 10% FCS and 1% BSA in PBST.

Samples were stained with primary antibody in blocking solution. Primary antibodies used:

Brachyury (custom-made, 1:200), Sox17 (R&D systems AF1924, 1:200), Foxa2 (Cell

Signaling 8186, 1:200; R&D system AF2400, 1:100). Secondary antibodies: DyLight

488/549/649 conjugated secondary antibodies (anti-mouse, anti-goat, or anti- rabbit,

1:400, Jackson ImmunoResearch). For triple staining, to avoid secondary antibody cross-

talk, Brachyury antibody was directly conjugated with DyLight 488 Amine-Reactive Dye

and Kits (Pierce). Nuclei were counterstained with DAPI. All imaging analysis was

performed by using ImageJ (11).

Genotyping of Tc/Tc mouse

Tc allele has a 19bp deletion from 1161 to 1179 bp of the Brachyury coding DNA

sequence (CDS), leading to a frame shift and substituting the C-terminal 50 amino acids

http://www.rndsystems.com/pdf/af1924.pdf


to an unrelated sequence (32 amino acids) (8). For genotyping, we performed genomic

DNA PCR by two primers flanking the deletion:

P1, 5-CTCTGGTCTGTGAGCAATGGTAC-3;

P2, 5-GTCAGAAATGTCTGTGACTGTAGCAG-3;

DNA sequencing was used to confirm the deletion. Genomic DNA was recovered by

TRizol LS reagent (Invitrogen) after Total RNA extraction.

mRNA-seq Library Preparation

Total RNA was extracted from EB and embryo samples by TRIzol LS reagent (Invitrogen).

For EB samples, 8µg of the total RNA from each sample was convert to mRNA-seq library

using mRNA-Seq Sample Prep Kit (Illumina). For mouse embryo samples, we used 4~5

Tc/Tc or WT embryos at each time point (E7.5~8.0, E10.0~10.5). mRNA was first

enriched from 2µg of the total RNA by Dynabeads Oligo(dT) (Invitrogen). Then, directional

mRNA-seq libraries was prepared by ScriptSe v2 Kits (Epicentre).

Expression Level Estimation, Differential Expression Testing and Gene Ontology

Analysis

We sequenced ESC and EB day 4 mRNA-seq samples in 50bp paired-end format (PE

reads; 1 lane HiSeq per sample). Mouse embryo directional RNA-seq samples were

sequenced in 50bp single-end format (1 lane HiSeq per sample). We mapped our

sequencing data back to the mouse reference genome (mm9) by Bowtie (12). Transcript

isoforms were reconstructed from PE reads using Tophat (13) and abundances estimated

using Cufflinks (14). Read counts were tallied for each Ensembl annotated protein-coding


gene (Ensembl 61) incremented by 1 and differential expression tested using Cuffdiff

using all qualified samples. Gene Ontology (15) analysis was performed separately on

up-regulated and down-regulated genes at each time-point using GOSeq (16) with

Wallenius gene-length correction.

ChIP-seq and ChIP-exo library preparation

Chromatin Immunoprecipitation (ChIP) was performed according to (17) with minor

modifications. Briefly, cross-linked EB chromatin was sheared using Covaris S2 system

to a size range of 100bp ~ 400bp. Immunoprecipitation was conducted with either a

specific antibody or mock IgG conjugated Protein A (rabbit and guinea pig) or G (mouse)

Sepharose beads (GE Healthcare). The reverse cross-linking was performed at 70oC

overnight. After RNAse A and Proteinase K treatment, sample was deproteinized with

UltraPure Phenol: Chloroform: Isoamyl Alcohol (Invitrogen) and further purified with

Qiaquick PCR purification kit (Qiagen). DNA concentration was measured with Quant-

iT™ PicoGreen dsDNA Reagent (invitrogen). About 10ng of ChIP enriched DNA was

converted to ChIP-seq library using ChIP-seq DNA Sample Prep Kit (Illumina). ChIP-exo

library was prepared by following the published protocol with minor modifications (18).

Specifically, we adapted the SoLid sequencer adaptors/primers to make the final library

compatible with the illumina Tru-seq small-RNA system. The detailed primer information

is in Dataset S5.

ChIP-seq Peak Calling, Seq Region Definition and GREAT Analysis


We sequenced chromatin-IP and control libraries in 50bp single-end format for ChIP-seq

libraries (one lane of HiSeq per sample). Reads were mapped to the mouse genome

(mm9) using Bowtie (12) and peaks called using MACS (2.0beta; (19)) at an approximate

1% FDR (“bound regions”). We merged overlapping bound regions from both antibodies

to produce a set of 3160 regions. Bound-regions and annotation-terms association was

calculated by Genomic Regions Enrichment of Annotations Tool (GREAT) (20).

Evolution Conservation Analysis

Mouse genes with one or more Brachyury binding sites 10kb < the TSS were considered

as Brachyury target genes (Dataset S4). The list of 218 Zebrafish Brachyury homolog (Ntl)

target genes was obtained from the report by Morley et al. (21) and we further converted

zfin gene symbols to ensembl gene symbols (Dataset S4). Common genomic regions

bound by Xenopus Brachyury homolog (XBra) (Stage 11-12.5) detected in both ChIP-seq

replicates (22) were correlated with JGI v4.1 assembly gene annotations. Genes with one

or more XBra binding sites 10kb < the TSS were considered as XBra Target genes

(Dataset S4). Zebrafish and Xenopus target genes were converted to mouse homolog

genes by ensemble BioMart (23) and then correlated with mouse target gene information.

The results are listed in Dataset S4.

ChIP-exo Peak Calling and Bound-region Definition

We sequenced exo library in 50bp single-end format by using the illumina HiSeq platform.

After removal of the 3 prime most 14 bp which tend to have higher error rates, we mapped

our sequencing data back to the mouse reference genome (mm9) by Bowtie (12). We


further reduced the mapped read regions to single 5’-end point, which reflects the cross-

linking point between protein and DNA. The resulting cross-linking point distribution was

used to identify peaks on the forward (Left) and reverse (Right) strand separately using

the peak calling algorithm in GeneTrack (24). For bound-region calculation, we first

identified any pairs of left and right peaks that were located within 20bps to each other.

Then, we defined the window between the middle point of the left peak and that of the

right peak as the bound-region. Peak-pairing and bound-region calculation were

performed with R programming (scripts are available upon request). The resulting 11503

paired-peaks were defined as exo-peaks (11k dataset). Top 10% of exo-peaks (1k

dataset) were used for motif discovery.

Motif Discovery, Localization and EMSA assay

The de novo motif discovery was conducted by Gimmemotifs (25) using 20bp of

sequence flanking the midpoint of exo-peaks from the 1k dataset. 5 motifs were recovered

from the analysis and were matched to known transcription factor binding sequences by

Jaspar (26). The plots for local raw-read enrichment and color charts for motif sequences

were generated by customized R scripts. We further calculated motif preference

localization results by using the motif_localization_plots.py module in Gimmemotifs.

Finally, we took 50bp of sequence flanking the peaks in the 11k dataset for co-localization

analysis by using pwmscan.py module in Gimmemotifs and bedtools (27) (intersectbed).

Likewise, we used the same number of 50bp random genomic regions to calculate

background co-localization events. EMSA assay were performed with standard protocol.


Flag-tagged Brachyury protein was first over-expressed in 293T cells and then purified

for EMSA reactions.


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Supporting Information Appendix for · Supporting Information Appendix for ... Global differential gene expression analysis by mRNA-seq. ... Color chart representation of 20bp of