Post on 23-Mar-2020
Uslu et al – Supplementary Information
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Long-range enhancers regulating Myc expression are required for normal facial
morphogenesis
Veli V Uslu et al.
Supplementary Information
Supplementary Figure 1: Conserved organization of the 8q24 region in mouse and human.
Supplementary Figure 2: Deletion series to delineate the MNE region
Supplementary Figure 3: Duplications series to delineate the MNE region
Supplementary Figure 4: Expression of genes flanking the CL/P region.
Supplementary Figure 5: Transposon insertions do not induce expression changes
Supplementary Figure 6: Morphological and cellular differences between del(8-17) and WT
Supplementary Figure 7:Comparison of gene expression in the face of del(8-17) vs WT
embryos
Supplementary Figure 8: Reduced expression of blood-related genes in del(14-17) mice.
Supplementary Figure 9: Functional and genetic structure of the human CL/P risk interval.
Supplementary Figure 10: Molecular nature of the 8q24 CL/P risk factor.
Supplementary Figure 11: RNA quality control and qPCR primer efficiencies.
Nature Genetics: doi:10.1038/ng.2971
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Supplementary Table 1: List of the different insertions spanning the CL/P region.
Supplementary Table 2: List of the deletions and duplications spanning the CL/P region.
Supplementary Table 3: Regions enriched for H3K27ac and H3K4me1 (.xls)
Supplementary Table 4: Mis-expressed genes in del(8-17) versus WT mice (.xls)
Supplementary Table 5: RNA-Seq data for the genes surrounding the MNE (.xls)
Supplementary Table 6: GO terms enrichment analysis for differentially expressed genes.
Supplementary Table 7: GO terms enrichment analysis for highly but differentially expressed
genes.
Supplementary Table 8: List of deregulated genes (p<0.05) involved in cranio-facial
development.
Supplementary Table 9: genotyping primers used for the different mouse alleles
Supplementary Table 10: primers used for qRT-PCR experiments
Nature Genetics: doi:10.1038/ng.2971
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Supplementary Figure 1
Conserved organization of the 8q24 region in mouse and human.
Representation of the 8q24 interval (hg19, chr. 8: 127,200,000–131,500,000) from the UCSC
Genome Browser64 with the 640-kb CL/P risk interval boxed3. ENCODE tracks summarizing
regulatory and transcription activities (from seven cell lines) are shown65, as well as the score
of evolutionary conservation of the sequence (GERP track66). The paucity of gene annotation,
transcriptional activity (RNA-seq tracks) and promoter-associated chromatin marks
(H3K4me3) highlights the ‘gene desert’ constituted by this region between PVT1 and
GSDMC. The region comprises, however, many evolutionarily conserved elements (peaks in
the GERP track) and potential tissue-specific enhancers (peaks in the H3K4me1 and
H3K27ac tracks). The Mouse Net track shows the extensive syntenic chain linking mouse and
human orthologous sequences, with extreme conservation in sequence and relative order
between the two species.
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Supplementary Figure 2 Deletion series to delineate the MNE region. (a) Schematic
representation of the different deletions (red bars) generated and analyzed along the interval,
with the different regulatory regions identified (blue, medionasal enhancer (MNE); orange,
nasal epithelial enhancer (NEE)) shown as ovals. (b–e) LacZ staining of E11.5 embryos with
different deletions, highlighting the persistence or loss of the two expression domains (blue
arrowhead, MNP; orange arrowhead, NC). Insets in c–e, 150-µm vibratome sections through
the head of embryos, showing strong expression in the nasal epithelium of del(8–14)
heterozygous embryos (c). This domain of staining is absent in del(14–15) embryos (d) and
weak but present in del(15–17) embryos (e).
Nature Genetics: doi:10.1038/ng.2971
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Supplementary Figure 3 Duplications series to delineate the MNE region. (a) Schematic
representation of the positions and LacZ expression patterns in E11.5 embryos for the 10a,
13a and 20a transposon insertions. Regulatory regions are indicated as before. The
topological boundary found around the Gsdmc cluster67, which overlaps with the regulatory
transition between the different landscapes, is shown with double red brackets. (b) Schematic
representation of the trans-allelic Cre-mediated recombination51 used to produce the different
duplications, as a reciprocal product of the deletions. (c) Representation of the different
duplications and (d) associated LacZ expression in E11.5 embryos. Duplications
encompassing the region (10–13) led to expression in the fronto- and medionasal processes,
whereas a duplication of the region (13–20) conferred expression in the nasal epithelium only.
Even though it is unclear whether topological boundaries are fully respected in the context of
rearrangements68, the different expression of the LacZ sensor for the dup(10–20) and dup(13–
20) alleles, which place it at the same distance from the centromeric CL/P region (blue oval),
can be better explained by the contribution of enhancer elements lying in the duplicated
telomeric regions.
Nature Genetics: doi:10.1038/ng.2971
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Supplementary Figure 4 Expression levels of the genes flanking the CL/P region in the face
of E11.5 embryos. Expression levels were measured by qRT-PCR and are shown with the
lowest expression levels (for Gsdmc) set as 1 (log10 scale). Error bars represent ± s.d. from
four independent biological replicates. *, the primers used cannot distinguish the different
tandemly duplicated Gsdmc genes
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Supplementary Figure 5 Transposon insertion does not induce expression changes.
Endogenous gene expression in the face of E11.5 embryos homozygous for expression
showing the strongest LacZ expression is not different from wild-type control. Expression
was determined by qRT-PCR (three biological replicates). Expression levels were normalized
to Gusb levels between samples and, for each gene, represent with wild-type levels equal to 1.
Error bars are ± s.d.
Nature Genetics: doi:10.1038/ng.2971
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Supplementary Figure 6 Morphological and cellular differences between del(8-17) and WT
(a) Comparison of different bone lengths and skull measures (IOD, interorbital distance;
NBL, nasal bone length; FBL, frontal bone length; PBL, parietal bone length) in 5-week-old
(n = 4 (del(8–17); n = 4 (wild-type)) mice. Del(8–17) mice showed reduced nasal and frontal
bone lengths (Student’s t test, P = 0.00398 and P = 0.00099, respectively). Boxplots show
median, 1st and 3rd quartiles. Whiskers indicate min./max (b) Cell proliferation in the face of
del(8–17) and wild-type E11.5 embryos. Mitotic cells were identified by staining for
phosphorylated H3 and counted on serial sections. Each dot represents the normalized
proportion of cells positive for phosphorylated H3 for a given section. Del(8–17) embryos
showed slight but significant differences (Student’s t test, P =1.77 × 10–6). Boxplots show
median, 1st and 3rd quartiles. Whiskers indicate 1.5 IQR of the 1st and 3rd quartiles. ***
indicates P <0.005
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Supplementary Figure 7 Expression changes in the face of del(8-17) embryos compared to
WT controls. (a) A heat map showing normalized expression values for all genes with a
minimum expression of 100 reads (summed across all samples). Each row corresponds to 1 of
the 13,586 genes under consideration, and the columns correspond to the different samples
(black, wild type; gray, deletion). Colors show gene expression on the log2 scale (blue, low
expression; yellow, high expression). (b) A heat map showing normalized expression values
for differentially expressed genes. Each row corresponds to a differentially expressed gene,
and columns correspond to the different samples (black, wild type; gray, deletion). Colors
show gene expression on the log2 scale (blue, low expression; yellow, high expression).
Nature Genetics: doi:10.1038/ng.2971
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Supplementary Figure 8 Reduced expression of blood-related genes in del(14-17) but not
del(8-14) mice. Several genes with restricted expression in blood cells had downregulated
expression in del(8–17) versus wild-type face samples. Overall, their expression levels were
low, consistent with the presence of a few small blood vessels in the dissected facial
mesenchyme. qPCR analysis of expression changes for some of these genes shows that this
misexpression is associated with another regulatory region, located in (14–17) and therefore
distinct from the MNE. **P < 0.01, *P < 0.05, Student’s t test. Error bars are ± s.d.
Nature Genetics: doi:10.1038/ng.2971
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Supplementary Figure 9 Genetic and functional organization of the CL/P interval on 8q24.
(a) Schematic representation of the 8q24 region, from the UCSC browser. The interval
showing strong association with CL/P identified by Birnbaum and colleagues3 is outlined in
red, with the position of the SNP (rs987525) with the lowest P value indicated by a red bar.
This interval consists of multiple LD blocks (HapMap Phased LOD track). Importantly,
multiple SNPs along this broad interval showed association with CL/P, in part independently
of rs987525 (refs. 3,5). The orthologous region to the (10–13) MNE is outlined in blue, with
ovals showing candidate enhancer modules in the region, including the Vista hs1877
element14. (b) The critical MNE region contains two main LD blocks, as shown by
Haploview, using HapMap CEU data (phase 2, r24)69.
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Supplementary Figure 10 – Molecular nature of the 8q24 CL/P risk factor
The 8q24 CL/P risk interval is a remote regulatory region (MNE) that specifically controls the
high levels of expression of MYC in the developing medionasal region. Genetic variation in
the MNE may perturb the GRN controlling the fate of the neural crest–derived mesenchymal
cells, possibly through NR2F1 and TFAP2A, and may alter the growth and metabolic potential
of the medial nasal process. This imbalance may be exacerbated by environmental (or
genetic) conditions, leading to defective fusions of the different facial processes.
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Supplementary Figure 11. RNA quality control and Primer Efficiency (a) RNA quality
measured by Bioanalyzer. RNA Integrity Number (RIN; value assigned from 0 to 10) was
calculated with Agilent 2100 Bioanalyzer software. Example histograms for three samples are
shown, and the minimum RIN value of the samples used for qRT-PCR was 9.10. (b) Primer
efficiency was measured using four- to eightfold dilutions of the cDNA stock. Curves show
log2 values for the dilution ratio plotted against Ct values from qRT-PCR amplification using
the different primer pairs.
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Supplementary Table 1 – List of the different insertions spanning the CL/P region.
Line_name Short Name Chr Position
Expression in E11.5 MNP LoxP Transposon transposon parent
196231
15 60133316 0 minus SB9 179039 197658e12 *
15 60550577 0 minus SB9 179039
205880 c8a 15 61051281 0 minus SB9 196231 or 194578 209545
15 61350326 0 minus SB9 194578
196895e2 * 1a 15 61940681 ++ minus SB9 179039 211151
15 62073790 + plus SB9 194578
194578
15 62168343 ++ plus SB9 179039 193642
15 62237968 ++ plus SB9 184347
197272
15 62461203 0 plus SB9 184347 188150 7a 15 62561825 ++ minus SB9 179039 193284
15 62614447 +++ minus SB9 179039
194832
15 62646407 +++ plus SB9 184347 190909 8b 15 62668503 ++ minus SB9 184347 184347 8a 15 62668548 +++ plus SB9 179039 196919
15 62668549 +++ minus SB9 184347
197662e6 *
15 62742085 ++ plus SB9 184347 193970 10a 15 62853768 0 minus SB9 184347
210632e2 *
15 62882596 ++ plus SB9 193970 194575 13a 15 63135562 +++ minus SB9 179039 195052 14a 15 63167953 ++ minus SB9 179039 195964
15 63181305 ++ minus SB9 179039
193058 14b 15 63185343 ++++ plus SB9 179039 193637 14c 15 63196469 ++ plus SB9 179039 192339 15a 15 63291835 ++++ plus SB9 179039 193315
15 63461422 + minus SB9 179039
196554
15 63523860 ++++ plus SB9 179039 194577
15 63547655 ++++ plus SB9 179039
179039 17a 15 63550550 ++++ plus SB9 176598 192857 17b 15 63550550 ++++ minus SB9 179039 186894
15 63562249 +++ plus SB9 179039
196337
15 63577759 + minus SB9 179039 191058-‐emb16 *
15 63595268 0 plus SB9 179039
192566 18a 15 63616937 0 plus SB9 179039 195308
15 63694045 0 minus SB9 179039
vu-‐emb1-‐12 *
15 63736919 0 minus SB9 179039 196896e9 *
15 63824158 0 minus SB9 179039
192331 20a 15 63831346 0 minus SB9 179039 192571
15 63937869 0 plus SB9 179039
180206 21a 15 63942706 0 minus SB9 SBlac-‐E
Positions are given based on genome assembly MGSCv37/mm9. Expression in the medio-nasal process (MNP) is determined by the relative intensity of the LacZ staining observed for the corresponding insertion (0: no expression; + to ++++: faint to very strong). Insertions assessed directly as F0 (for which ones only one embryo was obtained) are indicated with a star (*). All others were established as lines and multiple F1 embryos produced by mating transgenic males with wild-type females. Spatial distribution and intensity of the LacZ staining is reproducible amongst littermates and between different litters of the same insertion. Additional information available online with the TRACER database (tracerdatabase.embl.de)
Nature Genetics: doi:10.1038/ng.2971
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Supplementary Table 2 – List of the deletions and duplications spanning the CL/P region.
Line Cen breakpoint Tel breakpoint Region spanned (mm9) Length (bp)
del(c8-‐7) 205880 188150 chr15:61051281-‐62561825 1510544
del(7-‐10) 188150 193970 chr15:62561825-‐62853768 291943
del(8-‐14) 184347 193058 chr15:62668548-‐63185343 516795
del(7-‐14) 188150 195052 chr15:62561825-‐63167953 606128
del(8-‐17) 184347 179039 chr15:62668548-‐63550550 882002
del(13-‐20) 194575 192331 chr15:63135562-‐63831346 695784
dup(13-‐20) 194575 192331 chr15:63135562-‐63831346 695784
del(15-‐17) 192339 179039 chr15:63291835-‐63550550 258715
del(14-‐16) 195052 193315 chr15:63167953-‐63461422 293469
del(17-‐21) 192857 180206 chr15:63550550-‐63942706 392156
del(14-‐17) 193637 179039 chr15:63196469-‐63550550 354081
del(14-‐15) 193058 192339 chr15:63185343-‐63291835 106492
dup(10-‐20) 193970 192331 chr15:62853768-‐63831346 977578
dup(8-‐21) 190909 180206 chr15:62668503-‐63942706 1274203
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Supplementary Table 3: Regions enriched for H3K27ac and H3K4me1 :
Supplementary Excel file
Supplementary Table 4: Mis-expressed genes in del(8-17) versus WT mice (p-value
<0.05)
Supplementary Excel file
Supplementary Table 5: RNA-Seq data for the genes surrounding the MNE
Supplementary Excel file
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Supplementary Table 6 – Analysis of enrichment of GO terms in differentially expressed genes upon deletion of (8-‐17)
GO term Description P-value FDR q-value Enrichment (N, B, n, b)
GO:0044391 ribosomal subunit 3.99E-19 4.87E-16 45.07 (9493,74,37,13) GO:0003735 structural constituent of ribosome 8.19E-15 2.55E-11 34.00 (9493,83,37,11) GO:0022625 cytosolic large ribosomal subunit 1.42E-13 5.77E-11 105.65 (9493,17,37,7) GO:0005840 ribosome 2.73E-13 8.35E-11 24.98 (9493,113,37,11) GO:0006412 translation 1.57E-13 1.57E-09 17.28 (9493,193,37,13) GO:0003723 RNA binding 7.23E-08 7.51E-05 3.93 (9493,1176,37,18) GO:0019538 protein metabolic process 2.07E-05 2.29E-02 2.57 (9493,1900,37,19) GO:0050767 regulation of neurogenesis 2.40E-05 2.39E-02 6.45 (9493,318,37,8) GO:0032268 regulation of cellular protein metabolic
process 4.86E-05 4.04E-02 3.67 (9493,839,37,12)
GO:0051246 regulation of protein metabolic process 1.15E-04 4.97E-02 3.36 (9493,916,37,12) GO:0042127 regulation of cell proliferation 1.57E-04 5.81E-02 3.88 (9493,662,37,10) GO:0045595 regulation of cell differentiation 2.86E-04 8.64E-02 3.60 (9493,712,37,10) GO:2000026 regulation of multicellular organismal
development 3.13E-04 9.18E-02 3.56 (9493,720,37,10)
GO:0031325 positive regulation of cellular metabolic process
3.73E-04 9.55E-02 2.78 (9493,1200,37,13)
GO:0032270 positive regulation of cellular protein metabolic process
3.53E-04 9.76E-02 4.40 (9493,467,37,8)
GO:0008284 positive regulation of cell proliferation 4.03E-04 1.00E-01 5.00 (9493,359,37,7) GO:0009653 anatomical structure morphogenesis 4.54E-04 1.05E-01 3.40 (9493,754,37,10 GO:0009893 positive regulation of metabolic process 6.37E-04 1.35E-01 2.63 (9493,1267,37,13)
Analysis was performed with Gorilla70, by comparing differentially expressed genes with expression range
>100 and p-‐adj. <0.05 to genes detected with the same expression range in the tissue. Enrichment of GO
terms is calcuted as (b/n) / (B/N), with N = total number of genes; B = total number of genes associated
with a specific GO term, n=number of differentially expressed genes b=number of differential expressed
genes associated with a specific GO term. GO terms supported by less than 6 differentially expressed genes
and redundant ones were removed.
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Supplementary Table 7 – Analysis of enrichment of GO terms for highly expressed genes differentially expressed upon deletion of (8-‐17).
GO term Description P-value FDR q-value Enrichment (N, B, n, b) GO:0006412 translation 1.76E-15 5.77E-12 16.00 (384,18,16,12) GO:0019538 protein metabolic process 2.84E-06 1.86E-03 3.18 (384,98,16,13) GO:0009059 macromolecule biosynthetic process 3.24E-06 1.77E-03 3.15 (384,99,16,13) GO:0044267 cellular protein metabolic process 6.84E-06 3.20E-03 3.35 (384,86,16,12) GO:0044249 cellular biosynthetic process 1.57E-05 6.42E-03 2.79 (384,112,16,13) GO:0006417 regulation of translation 7.16E-05 2.61E-02 6.86 (384,21,16,6) GO:0071704 organic substance metabolic process 3.38E-04 1.01E-01 1.63 (384,236,16,16) GO:0043170 macromolecule metabolic process 4.57E-04 1.25E-01 1.77 (384,203,16,15) GO:0008152 metabolic process 9.99E-04 2.52E-01 1.52 (384,252,16,16) GO:0003735 structural constituent of ribosome 4.69E-16 3.67E-13 24.00 (384,10,16,10) GO:0005198 structural molecule activity 4.70E-09 1.84E-06 8.57 (384,28,16,10) GO:0003723 RNA binding 1.94E-05 5.07E-03 2.20 (384,164,16,15) GO:0044391 ribosomal subunit 8.92E-17 4.45E-14 22.00 (384,12,16,11) GO:0005840 ribosome 5.09E-15 1.27E-12 21.82 (384,11,16,10)
Analysis was performed with Gorilla70, by comparing differentially expressed genes with expression range
>3000 and p-‐adj. FDR<0.05, to genes detected with the same expression range in the tissue. Enrichment of
GO terms is calcuted as (b/n) / (B/N), with N = total number of genes; B = total number of genes
associated with a specific GO term, n=number of differentially expressed genes b=number of differential
expressed genes associated with a specific GO term. GO terms supported by less than 6 differentially
expressed genes and redundant ones were removed.
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Supplementary Table 8:
Deregulated genes (p<0.05) involved in cranio-facial development
Gene name
Expr. Change (log2)
Pval Pathway Relevance for CL/P and facial morphology references
Myc -‐2.583963 1.21275E-‐86 8q24 GWAS this work and ref 3
Etv5 0.2663346 1.78193E-‐05 Fgf 71
17693063 Bmp7 0.2623349 2.41086E-‐05 Bmp CL/P in mouse ko 72 Nr2f1 -‐0.2455414 2.67368E-‐05 NCC
20
Pdgfc 0.2006095 0.00015968 Pdgf mouse mutant, human linkage/GWAS 73,74
Pvrl4 0.485702 0.000170635 paralogous to CL/P-‐causing gene Pvrl1 75 Fzd6 0.3001039 0.000260691 Wnt Dusp4 0.3562477 0.000378409 Fgf Bmp4 0.2712896 0.000462893 BMP mutation causes CL in human
(epithelium) 76
Tgfa 0.3216962 0.000685507 human CPO 77
Lrp4 0.2686782 0.000782122 Wnt Col17a1 0.4056272 0.00269577 normal facial morphology GWAS 24 Dusp6 0.2163654 0.002969277 Fgf maxillary/mandibulary growth 78
Wnt5a 0.1963515 0.003162661 Wnt facial dysmorphism (dominant Robinow syndrome)
79
Eif3e -‐0.171818 0.0037729 translation NCC -‐ zebrafish 80 Spry4 0.282755 0.005827626 Fgf Twist1 0.1482429 0.006090016 NCC severe craniofacial defects in ko mice 81 Trp63 0.1432408 0.006184221 craniofacial defects, epithelium 82 Sp8 0.2064278 0.00652463 craniofacial centers 83
Dlx3 0.2222632 0.006984368 mild craniofacial phenotype
(mouse knockout) 84
Col9a1 -‐0.2032272 0.007139884 CP (Stickler syndrome) 85
Spry1 0.2737054 0.007458963 Fgf craniofacial defect incl. CP upon ectopic expression in NCC
86
Wnt9b 0.2672289 0.007815228 Wnt CL/P (mouse hypomorph) 34
Foxp1 -‐0.1272622 0.00849356 mild craniofacial abnormalities
in human 87
Tcf4 0.1165964 0.009933771 Wnt craniofacial defects in Tcf4/Lef1 ko mice 88
Rspo1 -‐0.2729092 0.01104947 Wnt 89
Fgf9 0.3517922 0.01288658 Fgf Rdh10 0.1974687 0.0131987 RA
90 Acvr2a 0.1388022 0.02026706 NCC in zebrafish 91 Bmp1 0.1320451 0.03430452 Bmp Cdh1 0.1435271 0.01860036 CL/P GWAS 92 Col2a1 -‐0.1306992 0.02858671 CP (Stickler syndrome) 85
10100048 Dkk1 0.373885 0.01742373 Wnt over-‐expression cause facial
malformations 88
Dlx6 0.2248032 0.01662266 89
Hmga2 0.1590215 0.01930564 normal facial morphology GWAS 93
Lef1 0.2186644 0.02763419 Wnt craniofacial defects in Tcf4/Lef1 ko mice 88
Msx1 0.1502287 0.03015016 NCC CP and craniofacial defects in mouse ko 94
Mtr -‐0.1142592 0.04963768 Folate metabolism
Snai2 0.1623218 0.02615497 NCC 95
Sox10 -‐0.3723462 0.04498194 NCC
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Genes from Supplementary Table 4 were annotated for reported function
(Fgf/Bmp/Wnt/retinoic acid signalling, NCC gene-regulatory network). Phenotypes
observed in mutant mice (gain or loss-of function) or in human patients carrying
mutations/polymorphism are briefly described.
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Supplementary Table 9 – Genotyping Primers
Insertion Name Primer sequence (L-‐end) Primer sequence (R-‐end)
Generic SB AAGTAGATGTCCTAACTGACTTGC TCCTAACTGACCTAAGACAGG Insertion specific Primers to use with the corresponding generic ones
196231 CCTGGAATCTCCTTTTGTTCAGG GTCCAACAGCTTGTCCAGATCC
205880 GGACGTTTTGTGCTGAGAAAGG TCCTGGAACCTTCTGAAACAGG
209545 TTCATGGCAGGTATGATTGTGG 211151 TCACCTGAGCAAGTCTGTCTCC ACAGGAGGACCCATTAAACACC
194578 CAGGAGTTTGCCAATCAACAGTG GAAAGCAAGTGGGGAAGTCAGAG
193642 TTCTCTGGGTTGGAAGCTGTG AATCGGCCCACAGTTCTGAAT
197272 CAACTCTCTGCTCCACTGATGC TGGTCTTGAAGCATCCTCTTCC
188150 ATGGTTGGCCAAAGAAGTTG AATGTGGCCACTCTCTTTGC
193284 GATAAGTTTCCTTCCCCCATCG CACATTAGTGCGACCCATTCAA
194832
CCTCTCATGTTGACAGTCAAGACG
190909 ATCCCATGAAAGGCATGGAGAG TGGTGTCTCTTCCCACCATTTG
184347 TGGTGTCTCTTCCCACCATTTG ATCCCATGAAAGGCATGGAGAG
196919 ATCCCATGAAAGGCATGGAGAG TGGTGTCTCTTCCCACCATTTG
193970 TGCTCAGTCCAGTGGATGACTATG TGGTTGCCTTTTTGTCTGATTGT
194575 CCACAAGTAGATCAGCCACAAACC ATTGTTGGCAAAACACAACAGG
195052 TTGGAATTTGAAAACGACATTGG GCAGTCTGCTTGTTTGTTTGTTTG
195964 AACCCCACTTCCTGAACCACTG TGTGTCACACTGGTGGAAAAGAAAC
193058 TTGGGTACATCTGTCACCAGAGTC TCAGAGTGTGGTCAACTGTGGAA
193637 GCATGGATTCTATGGGTGTTGG CCTCCTGGGATTTCCATGACTC
192339 AATGCCAAAGACAAGGACTCCAG GATGGGACTTCCCACATAACAGC
193315 GGCCTAGCAAACACAGAAGTGG ATCCACTCCCCTCTCTGTTTCC
196554 GGCCTAATCCCCTGTAATGACC AAGGGGGCTTGATTTGAATAGC
194577 CTTCACACTTGACAAGGGGTGTG TGTGTTTGGACACGGAAAATGAC
179039 GAGCAACGTGCTGATCTATGGG GTTCCTCCCAAGGTTCATGCTC
192857 GTTCCTCCCAAGGTTCATGCTC GAGCAACGTGCTGATCTATGGG
186894 CCTTGCCATTGTGTTCTGAG TGATGTGGTGACTGACATCTGA
196337 CAGCCTGACAGAAGAGAGAGACC TATCCATAAGGGATGGCAATGG
192566 CATAGCTCTGAGTGCCTCCAAAAG TGAGTATTTTTGCATCGATATCATAACA
195308 CCATGAGAGCTGGAGAGAGTCTTG AGTTATTGTCCGGTCAGGCAAAG
192331 CAGGAGGCTTTGGACTCAACACT CCTCTTTTGCCAACGTCTTCC
192571 ACCCTTGGCTGAAGACATACCA CAGGACTCCAGTCATGTGATGC
180206 GGCTTTGACCCTGACTTTAGG ATACCACCATGCTTGGCTTGAC
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Supplementary Table 10 – qPCR primers
qPCR Primer F Sequence (5’-‐3’) qPCR Primer R Sequence (5’-‐3’)
Csf-‐1 F GGAGCTCTGGGACCTGCTC Csf-‐1 R CTACGTCCCGGTGGATGC
ApoE F ACCCTGGAGGCTAAGGACTTG ApoE R TCATCTTCGCAATTGTGATTGG
Ano1 F AAGTAAACGGCGGAAGTGTGG Ano1 R CATAGTCCCCATCGTGCAGAG
Nr2F1 F CATCGTGCTATTCACGTCAGATG Nr2F1 R GATTTCTCCTGCAGGCTTTCG
Tpi1 F CTTCGTTGGGGGCAACTG Tpi1 R CGGTGCACAAACCACCTC
Itgb3 F ACACCAGTGGGAGGGCAGTC Itgb3 R TATCAGGACCCTTGGGACACTC
Sox11 F GGAGCTGAGCGAGATGATCG Sox11 R AACACCAGGTCGGAGAAGTTCG
A1bg-‐201 F TGGAGCTGCGGGTGAATG A1bg-‐201 R CCAGATGTACTGTGCTTTTCCAC
Fam84b F CCAGGGAAAGGATTCAATTAAGG Fam84b R CACAACAGCAGGCCAAAAACA
eGFP F GGGCACAAGCTGGAGTACAAC eGFP R CTGCTTGTCGGCCATGATATAG
cMyc F CCCTAGTGCTGCATGAGGAGACAC cMyc R CCACAGACACCACATCAATTTCTTCC
Pvt1 F CTGAGGTGGAGGAAGTTGCCCTTG Pvt1 R GGCCACCTCAATCAGGCAGTGTC
Asap1 F AAGAACGGGATCCTGACCATCTCC Asap1 R TGGCAGGTGAGGAGGTTTAACTTAGC
Gsdmc F GCAATCAAAGGGATCATCAACCAG Gsdmc R TGAATCTGTTTTCTCTGTTTGCCACTG
GusB F CTCTGGTGGCCTTACCTGAT GusB R CAGTTGTTGTCACCTTCACCTC
Pdhb F TGTTGTCCACTCCCTACCCTAGATAC Pdhb R CATTCTTATCTTGCCCCTTCCAGTG
Rplp1 -‐F CCTGGCTTGTTTGCCAAGG Rplp1 -‐R GCAGTGGATGGAGCAGCAC
Rps20 -‐F CCTGACTCACCGCTGTTCG Rps20 -‐R CGTCTTTCCGGTATCTTTAAATGC
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