www.sciencemag.org/cgi/content/full/1163802/DC1

Supporting Online Material for

The Air Noncoding RNA Epigenetically Silences Transcription by Targeting G9a to Chromatin

Takashi Nagano, Jennifer A. Mitchell, Lionel A. Sanz, Florian M. Pauler, Anne C. Ferguson-Smith, Robert Feil, Peter Fraser*

*To whom correspondence should be addressed. E-mail: peter.fraser@bbsrc.ac.uk

Published 6 November 2008 on Science Express

DOI: 10.1126/science.1163802

This PDF file includes

Materials and Methods SOM Text Figs. S1 to S8 Tables S1 to S5 References

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Materials and Methods

Mouse lines. We used Balb/c mice in this study unless stated otherwise. Wild type male or

female mice were mated to Thp heterozygotes to obtain both maternal (Thp/+) or paternal

(+/Thp) transmission of the Thp deletion (1). Air-T mutant mice (2) and G9a-/- mice (3) have

been described previously. For single strand conformation polymorphism (SSCP) analysis,

C57BL/6 females and M. musculus molossinus males (inbred stain JF1) were mated to

obtain F1 hybrid conceptuses. All experimental procedures using mice were carried out

under a project license granted by the Home Office UK, and a licence granted by the

‘Direction Départementale des Services Vétérinaires’, Montpellier, France.

Tissues. Placentas and embryos were removed from pregnant mice at E8.5, E9.5, E11.5 or

E15.5, and hearts were removed from adult mice. The tissues were immediately frozen and

stored at -80ºC until use. For RNA FISH, combined RNA/DNA FISH and RNA TRAP

experiments, 14 µm-thick frozen sections were made from frozen tissues on a cryostat and

mounted on silane-coated glass slides.

Quantitative RT-PCR expression analysis. Placentas carrying maternal- or paternally-

derived Thp deletion were identified by DNA methylation analysis of the Air and Igf2r

promoters. Frozen placentas were crushed to a fine powder in a mortar and pestle on dry

ice. Crushed tissue was homogenised for 1 min on ice in 5ml RNA-Bee (AMS

Biotechnology) per placenta. Total RNA was extracted according to the manufacturer’s

specifications, treated with 10U DNase I (Roche) for 30 minutes at 37˚C and purified by

phenol/chloroform extraction and ethanol precipitation.

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Reverse transcription (RT) was carried out on 4 µg of RNA with random

hexanucleotide primers (5 ng/µl final concentration, GE Healthcare) using superscript II

(Invitrogen) according to the manufacturer’s protocol. RT negative controls were prepared

and treated identically except that the reverse transcriptase enzyme was omitted from the

reaction. After the RT reaction was completed the sample was diluted to 100 µl with

molecular biology grade water. Real-time PCR was performed with an ABI PRISM 7000

Sequence Detection System using SYBR green PCR Master Mix (Applied Biosystems). 1

µl of cDNA was used for each real-time PCR reaction which were performed in triplicate,

with the following thermal cycling conditions: 50˚C for 2 minutes and 95˚C for 10 minutes,

followed by 40 cycles of 95˚C for 15 seconds and 62˚C for 1 minute. The relative amount

of cDNA amplified for each primer pair was calculated by comparing to a genomic DNA

standard curve. For primer sequences, see Supplementary Table S4.

RNA FISH. The sections were briefly air-dried, extracted with 0.5% Triton X-100 in

phosphate-buffered saline (PBS) on ice, fixed in 4% formaldehyde, 5% acetic acid for 18

min at room temperature, washed 3 times in PBS for 5 min each, dehydrated in 70-100%

ethanol series and air-dried. We carried out probe hybridization and subsequent procedures

as described previously (4). We visualized Air and Slc22a3 transcripts with antisense

dinitrophenol- and digoxigenin-labelled single-stranded DNA probes, respectively, which

were prepared as described (4). Our probe for Air corresponded to nt 120875-123719 of

GenBank AJ249895, the probe for Slc22a3 to nt 4971942-4974278 of GenBank

NT_039638 and the probe for Igf2r to nt 124243-126190 of GenBank NT_097336.1. Since

our Slc22a3 and Igf2r probes were directed to intron 1 and 2, respectively, they only

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recognize nascent transcripts. For Slc22a3 and Igf2r allelic transcription analyses we

counted a minimum of seventy nuclei with Air signals, which marked labyrinthine

trophoblasts.

RNA/DNA FISH. Probe labelling, pre-hybridization treatments, hybridization, post-

hybridization wash and signal detection for RNA FISH were carried out as described

above. After RNA FISH, the sections were fixed again in 4% formaldehyde in PBS for 30

min at room temperature, washed 3 times in PBS for 5 min each, treated with 100 µg/ml

RNase A in 2 × SSC for 1 h at 37ºC. After a brief rinse in 2 × SSC, the sections were

denatured in 70% formamide, 2 × SSC for 5 min at 85ºC, dehydrated with 70% ethanol for

5 min at -20ºC followed by 100% ethanol for 3 min on ice, then air-dried before

hybridization with DNA FISH probe. We carried out probe preparation, hybridization, post-

hybridization wash and signal detection for DNA FISH as previously described (5). A 170

kb BAC clone RP24-240D2 from BACPAC Resources, labelled with biotin by nick

translation, was used as a DNA FISH probe to detect the Slc22a3 locus.

Image analysis. To calculate the cover index, z-stack images of each pair of FISH signals

were captured with a Zeiss LSM510 META confocal microscope, and the cover index was

calculated using Volocity software (Improvision). To determine the cover index, the

Slc22a3 signal centre was first identified as the brightest three pixels (one pixel

corresponded approximately to 0.05 µm × 0.05 µm on tissue sections) among the Slc22a3

signal of the pair. Then the cover index was calculated as a ratio of the average Air signal

intensity at the Slc22a3 signal centre divided by the average of the three brightest Air signal

intensities within the pair.

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RNA TRAP. RNA TRAP is derived from RNA FISH and uses the molecular targeting

power of in situ hybridization, to covalently tag chromatin in the immediate vicinity of a

specific RNA in the nucleus (4, 6). Briefly, horseradish peroxidase-conjugated (HRP)

antibodies were directed to the site of the hapten-labelled Air RNA FISH probe on placental

tissue sections (fig S3A). The HRP then catalyzes the covalent deposition of a biotinyl-

tyramide on chromatin and other proteins in the immediate vicinity of the RNA. We

verified biotinyl-tyramide labelling to a single site per nucleus in the labyrinthine

trophoblast layer of the placenta using fluorescently conjugated streptavidin (fig S3B). The

sections were then removed from the slides and sonicated to fragment chromatin to an

average size of < 500 base pairs (fig S3C and fig S4). Biotin-labelled chromatin fragments

were recovered on streptavidin beads (fig S3D) and assessed for enrichment of specific

sequences by real-time PCR.

We used 500-675 tissue sections for each experiment. Probe preparation, pre-hybridization,

hybridization and post-hybridization washes were carried out as described (4). All the

subsequent procedures were at room temperature unless stated otherwise. Endogenous

peroxidases were quenched in 3% H2O2 in PBS for 60 min, washed in PBS 3 times and

TST (100 mM Tris pH7.5, 150 mM NaCl, 0.05% Tween 20) for 5 min each, and blocked

with TSB (100 mM Tris pH7.5, 150 mM NaCl, 0.5% blocking reagent (PerkinElmer) for

30 min. We incubated slides with anti-digoxigenin antibody conjugated with horseradish

peroxidase (Roche) diluted 1:100 in TSB for 30 min in a humidified chamber, washed

twice for 5 min each in TST and then incubated them for 10 min with biotinyl-tyramide

(PerkinElmer) diluted 1:50 with TSA diluent (PerkinElmer) containing 20% sodium

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dextran sulfate. The slides were then quenched again in 0.5% H2O2 in PBS for 10 min,

washed twice in TST for 5 min each and transferred to 100 mM Tris pH 7.5, 150 mM NaCl

on ice for scraping.

We checked biotin deposition by staining 2-3 sections with a FITC-conjugated

streptavidin (Molecular Probes; diluted 1:200 in TSB) for 30 min. The sections were then

washed, dehydrated, mounted and observed under the fluorescent microscope.

We scraped the rest of the sections from the glass slides in 10 mM Tris pH 8.0,

10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100 on ice. The sections were then spun

down at 2,900g for 25 min at 4ºC, resuspended in ice-cold 10 mM Tris pH 8.0, 100 mM

NaCl, 1 mM EDTA, 0.5 mM EGTA and sonicated on ice 3 times for 5 min each (with 5

min interval on ice between bursts) using a Microson Ultrasonic Cell Disruptor set at level

3. We centrifuged the material for 15 min at 10,000g at 4ºC and obtained the supernatant

containing the soluble chromatin. This procedure routinely yielded chromatin fragments

less than 1 kb (see fig. S2). We set aside 10% of the soluble chromatin as the input fraction,

and mixed the rest with 250 µl of streptavidin agarose beads (Molecular Probes) in RIPA

buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate,

0.1% sodium dodecyl sulfate, 1 × Complete protease inhibitor cocktail (Roche) at 4ºC with

constant agitation overnight. After binding, we washed the streptavidin agarose beads 3

times with 1 ml PBS at 4ºC for 30 min each. We then reversed the formaldehyde crosslinks

and purified DNA from the streptavidin agarose beads (eluted in 70 µl as pull down

fraction) according to manufacturer’s instructions using QIAamp DNA micro kit (Qiagen).

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The input fraction was processed via the same method as the pull down fraction for

crosslink reversal and purification, and eluted in 100 µl.

The enrichment of genomic DNA in pull down versus input fractions was

analyzed by real-time PCR using the primer pairs shown in Supplementary Table S5. Real-

time PCR was carried out with an ABI PRISM 7000 sequence detector using 2 × SYBR

green PCR master mix (Applied Biosystems). For each primer pair, we generated a

standard curve using 1:200, 1:500, 1:1250 and 1:2500 dilution of the input fraction, which

was then used to quantify the concentration in the pulldown fraction. We performed three

biological replicates for each time point and tissue and all PCR reactions were done in

triplicate. We analyzed the melting curve of all PCR products to ensure that all reactions

gave a single product.

Chromatin Immunoprecipitation to analyze histone modification. After careful

dissection to remove the fetal membranes and maternal decidua, placentas were flash

frozen in liquid nitrogen. Heterozygous placentas (Thp/+ or +/Thp) were crushed to a fine

powder in a mortar and pestle on dry ice. Native ChIP was performed following the

protocol detailed in (7, 8), using the following antibodies: H3K4me3 (Abcam ab8580),

H3K27me3 (Abcam ab6002), H3K9me3 (Abcam ab8898), H3K4me2 (Upstate 07-030),

rabbit anti-Goat (Sigma G4018; as a negative control). Input and immunoprecipitated

samples were quantified using a spectrophotometer (NanoDrop). Real-time PCR was

performed with an ABI PRISM 7000 Sequence Detection System using SYBR green PCR

Master Mix (Applied Biosystems). The real-time PCR reactions were carried out in

triplicate with the following thermal cycling conditions: 50˚C for 2 minutes and 95˚C for

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10 minutes, followed by 40 cycles of 95˚C for 15 seconds and 62˚C for 1 minute. The

relative enrichment for each primer pair was calculated by comparing to a standard curve of

the input and normalising to the 2.5 Mb upstream primer pair, which showed a consistent

level of enrichment for all modifications tested. For primer sequences, see Supplementary

Table S5.

RNA Immnoprecipitation (RNA IP). E11.5 placentas were crushed to a fine powder in a

mortar and pestle on dry ice. Cross-linking and lysis were done as described (9), followed

by sonication on ice 3 times for 15 sec each (with 1 min 45 sec interval on ice between

bursts) using a Microson Ultrasonic Cell Disruptor set at level 3. The lysate was

immunoprecipitated with 10 µg of either anti-G9a antibody (R&D Systems PP-A8620A-00

or Upstate 07-551) or appropriate control IgG of the same isotype (Sigma M5409 or Abcam

ab27478) and 100 µl of Dynabeads protein A (Invitrogen). After washing with PBS twice

at 4˚C, samples were treated with 20 U of RQ1 DNase (Promega) for 30 min at 37˚C before

elution by adding NaHCO3 (final 0.1 M) and SDS (final 1%). Then RNA was extracted

using TRI Reagent LS (Sigma) and reverse transcribed with Air-specific primer (5'-

TTAAGCATCTCTGGGCTCTTCTC -3') and AffinityScript multiple temperature reverse

transcriptase (Stratagene) at 55˚C. The cDNAs from pull down fractions were quantified by

real-time PCR as above using the following primer pair (5'-

CACATTACTCTCGCTCATGTGTC -3' and 5'- GGAAGGGACGTTCTGTATCTCT -3';

amplicon located approximately 61 kb from Air 5' end) and genomic DNA standard curve.

Chromatin Immunoprecipitation to analyze G9a association. Sample cross-linking,

lysis, sonication (4 times for 15 sec each), immunoprecipitation (with antibodies above) and

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washes were done essentially as described (9), and immunoprecipitates were eluted with

1% SDS, 0.1M NaHCO3. Cross-links were reversed with 0.5% SDS and 100 µg/ml

proteinase K at 65˚C for overnight. After DNA purification, quantification of pull down

fraction was performed by real-time PCR as described above using primer pairs in

Supplementary Table S5 and input fractions with DNA standard curves.

Single strand conformation polymorphism (SSCP) assay. PCR-SSCP assay was carried

out as previously described (10). Breifly, total RNA was extracted from E8.5 or E9.5 F1

hybrid placentas and embryos from C57BL/6 (B6) female x M. molossinus male (inbred

stain JF1) cross. cDNAs were synthesized using random oligonucleotides and PCR

amplifications (35 cycles) were performed on the cDNAs in the presence of radio-active

33P-dCTP with the following primers (amplicon is in the 3' UTR of each gene) ; 5'-

GAGCATCACACTCCGTTCTCC-3' and 5'-AAGCCTTCTGGAGCAGAGAC-3' for

Slc22a3, 5'-GAGTGGCCGAGCTGAGCCAG-3' and 5'-GCAGTGTCGTACACCCAGCC-

3' for Slc22a2, 5'-GTGTGCTTCCAACAGGTGCCC-3' and 5'-

GGGTATGTAAATGCCACCTAT-3' for Igf2r. After PCR amplification, the PCR

products were denatured and run through a non-denaturing poly-acrylamide gel in order to

reveal single strand conformation polymorphisms (SSCPs) between maternally and

paternally derived transcripts.

Statistical analysis. In analyzing the cover index we employed Fisher's exact test. RNA

TRAP data were analysed by two-way ANOVA. Where appropriate, contrast tests were

applied to assess significant differences. These analyses were carried out using SPSS 14.0

(SPSS Inc). We used t-test to compare G9a association with maternal and paternal loci. To

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compare G9a association with the Slc22a3 promoter region in wild type, +/Air-T, Thp/+ and

+/Thp placentas, we used one-way ANOVA and Tukey's least significant difference. In

comparing Slc22a3 or Igf2r RNA FISH signals in trans versus in cis to Air signals in wild

type and G9a null placentas, we employed a t-test.

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Supporting text

We found allele-specific enrichments of H3K4 modifications at the maternal Igf2r and

paternal Air promoters (fig. S5), reflecting their allele-specific, imprinted expression, and

consistent with their H3K4 profiles in other cell types (11, 12). H3K4 methylation was also

present at the Slc22a3 promoter in placenta, however this modification was not allele

specific since both maternal and paternal alleles showed equivalent modifications at E11.5

that were reduced similarly at E15.5.

H3K9me3 enrichment was found at the maternal Air promoter in E11.5 and E15.5 placenta

(Fig. 3), consistent with other cell types (12). In addition, we detected H3K9me3

enrichment at the paternal Air promoter in E11.5 placenta (Fig. 3), which may reflect the

low level of Air expression in early placenta (see fig. S1).

A peak of histone H3K27 tri-methylation was found on both maternal and paternal Slc22a3

promoters at E11.5 placenta (fig. S5), consistent with previous chromatin

immunoprecipitation (ChIP) analyses using mouse ES cells (11). The bi-allelic nature of

the H3K27me3 mark in placenta suggests that this modification is not the result of Air

accumulation at the Slc22a3 promoter.

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Supporting Figures

Supporting fig. S1. Allele-specific RT-PCR analysis of steady state RNA and primary transcripts. Total RNA from +/Thp and Thp/+ placentas was quantified by real time RT-PCR. Upper panel shows data from primer pairs designed to exon sequences. Lower panel shows data from primer pairs to intron sequences. *Units represent ng of genomic DNA which show equivalent amplification. Error bars are ± SEM.

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Supporting fig. S2. RNA FISH dye-swap experiments on mouse placental tissue sections with probes that recognize Air and either Slc22a3 intron 1 or Igf2r intron 2. The image shown in the panel on the left is a dye-swap of the RNA FISH shown in Fig 1A. The middle and right panels are dye-swaps for Air and Igf2r. Each hybridization is visualized either in green (for digoxigenin-labeled probes) or red (for dinitrophenol-labeled probes) as indicated above. DAPI staining in blue. Scale bar, 5 µm.

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Supporting fig. S3. Schematic summary of RNA TRAP method. (A) Putative ncRNA (red) associated with chromatin is detected by in situ hybridization with RNA FISH probe (blue) labelled with digoxigenin (blue circles). Next, an anti-digoxigenin Fab fragment conjugated with horseradish peroxidase (HRP; yellow circles) is added to localize HRP activity to the site of the RNA. Upon contact with HRP the biotinyl-tyramide (orange circles) is activated and covalently attaches to electron-rich moieties (principally tyrosines) in the immediate vicinity. (B) Visualization of deposition of biotin on tissue sections with a FITC-conjugated streptavidin (green stars; see Fig. 2A). (C) Remaining tissue sections were scraped off the glass slides and fragmented by sonication (input fraction). (D) Biotinylated chromatin purified by affinity chromatography using streptavidin beads (pulldown fraction).

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Supporting fig. S4. Fragmentation of RNA TRAP DNA. Agarose gel electrophoresis of RNA TRAP DNA before and after sonication showing that chromatin is fragmented to an average size of <500 base pairs.

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Supporting fig. S5. Allele-specific ChIP analysis of histone modifications. Allele-specific H3K4 tri-methylation, H3K4 di-methylation and H3K27 tri-methylation within the locus were analyzed by ChIP using +/Thp and Thp/+ placentas at E11.5 and E15.5. The fold enrichment in the pull down to input fraction at each data point was normalized to the enrichment at 2.5 Mb upstream. Shown are the mean fold enrichment values ± SD from one biological replicate experiment. A biological replicate showed similar results. Schematic map of the locus is shown below with vertical dashed lines at the imprinted gene promoters.

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Supporting fig. S6. RNA IP in E11.5 placenta using a different anti-G9a antibody (Upstate) from the one used in Fig. 4A. Air RNA is detected by RT-PCR as in Fig. 4A. The y-axis value represents the mean ± SEM derived from several measurements in a single experiment. A biological replicate showed similar results.

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Supporting fig. S7. Allele-specific ChIP in Thp deletion placentas at E11.5 using an different anti-G9a antibody (Upstate) from the one used in Fig. 4B. The mean fold enrichment ± SEM of G9a-pull down over IgG-pull down, normalized to the corresponding pull down at 2.5 Mb upstream is shown.

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Supporting fig. S8. Allele-specific detection of Slc22a3, Slc22a2 and Igf2r transcripts in E8.5 and E9.5 F1 hybrid placentas and embryos from C57BL/6 (B6) female x M. musculus molossinus male (inbred stain JF1) cross. Each of the cDNAs was analyzed for single strand conformation polymorphisms (SSCPs) in the 3' UTR to determine the parental origin of the transcripts.

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Supporting table S1

Cover index comparisons. Shown are the percentage of Air / Slc22a3 signal pairs with cover index above or below 0.4. p values are shown for comparison between E11.5 RNA/DNA FISH and E15.5 RNA/DNA FISH (p = 0.016) and between E15.5 RNA/DNA FISH and E15.5 RNA FISH (p < 0.0001). p values determined by Fisher’s Exact Test.

cover index E11.5 placenta

RNA/DNA FISH

E15.5 placenta

RNA/DNA FISH

E15.5 placenta

RNA FISH

< 0.4 20.0% (7 / 35) 47.5% (19 / 40) 90.9 % (50 / 55)

≥ 0.4 80.0% (28 / 35) 52.5% (21 / 40) 9.1% (5 / 55)

p = 0.016

p < 0.0001

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Supporting table S2

List of p values from statistical analyses of RNA TRAP experiments comparing each genomic region with 2.3 Mb downstream from Air locus within each tissue

approximate

position

approximate

distance from Air

transcription start E11.5 placenta E15.5 placenta adult heart

upstream of Plg 382 kb

Plg promoter 363 kb p = 0.629

Plg intron 10 342 kb p = 0.004 p = 0.010

Plg intron 17 325 kb p = 0.005 p = 0.041

Slc22a3 intron 5 285 kb p = 0.024 p = 0.540

Slc22a3 promoter 234 kb p < 0.001 p = 0.036 p = 0.060

between Slc22a3

and Slc22a2 224 kb p = 0.073

between Slc22a3

and Slc22a2 204 kb

Slc22a2 promoter 157 kb p = 0.086

Slc22a2 exon 3 142 kb p = 0.091

between Slc22a2

and Slc22a1 103 kb

Slc22a1 intron 3 76 kb

Slc22a1 promoter 66 kb p = 0.035 p = 0.044

Igf2r intron 41 51 kb p = 0.117 p = 0.045

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Igf2r intron 9 16 kb

5' end of Air 2 kb p = 0.054

Igf2r promoter 29 kb

middle of Air 65 kb p < 0.001

3' end of Air 106 kb p = 0.004

Mas1 promoter 121 kb

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Supporting table S3

List of p values from statistical analyses of RNA TRAP experiments comparing across the three tissues at each genomic region.

approximate

position

approximate

distance from Air

transcription start

E11.5 vs.

E15.5 placenta

E11.5 placenta vs.

adult heart

E15.5 placenta vs.

adult heart

upstream of Plg 382 kb

Plg promoter 363 kb p = 0.048 p = 0.017

Plg intron 10 342 kb p = 0.05

Plg intron 17 325 kb p = 0.002 p = 0.023

Slc22a3 intron 5 285 kb p < 0.001 p = 0.039

Slc22a3 promoter 234 kb p < 0.001 p < 0.001

between Slc22a3

and Slc22a2 224 kb

between Slc22a3

and Slc22a2 204 kb

Slc22a2 promoter 157 kb p = 0.03

Slc22a2 exon 3 142 kb

between Slc22a2

and Slc22a1 103 kb p = 0.07

Slc22a1 intron 3 76 kb p = 0.054

Slc22a1 promoter 66 kb

Igf2r intron 41 51 kb

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Igf2r intron 9 16 kb p = 0.071 p = 0.084

5' end of Air 2 kb p = 0.037 p = 0.006

Igf2r promoter 29 kb

middle of Air 65 kb p = 0.005 p < 0.001

3' end of Air 106 kb p = 0.034 p = 0.085

Mas1 promoter 121 kb

2.3 Mb

downstream

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Supporting table S4

List of primers used for real-time RT-PCR analyses for steady state RNA and primary transcripts.

approximate

distance of

the PCR

product from

Air start site*

approximate

position of

the PCR

product

primer 1 sequence

(5' to 3')

primer 2 sequence

(5' to 3')

236 kb

upstream

Slc22a3 exon 1 GGAAGCCACTAATACCAGCTCAG ATGGTAGAGTGGGTCTCCACATAG

142 kb

upstream

Slc22a2 exon 3 GTCACCATCCTCATCAATGCTATC ACCAGTCCCTGTAGAAATCGAAAC

27 kb

upstream

Igf2r exon 19 TAGAGTATGTGAACGGCTCTGCTT ATAAATCCTCGACCACAGACAAGA

65 kb

downstream

middle of Air ACTTTGACAGAACAATCGGCTCAG GAACATTTGCAAAGGACAGTCGAG

248 kb

upstream

Slc22a3 intron 1 CTCACAGATAGCAAAGGGAGAAC AGGACTGAGCTTGACAGAAGAAC

157 kb

upstream

Slc22a2 intron 1 GTGCTCCGCTAAGGAGTATTAT CATGGTCTAACCTATAGCCTCA

16 kb

upstream

Igf 2r intron 9 CTTAAGCTGCTAGTTGGCTTCACA CGTCCAAAACCACCATCTACAAG

*Based on Ensembl mouse Release 41.

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Supporting table S5

List of primers used for real-time PCR in RNA TRAP and ChIP experiments.

approximate

distance of

the PCR

product from

Air start site*

approximate

position of

the PCR

product

primer 1 sequence

(5' to 3')

primer 2 sequence

(5' to 3')

~ 2.5 Mb

upstream

Qk intron 2 ATAAGAAGGTGACGGTCTCTGTCC CTTCCTGCTCACCTGTAAGTTCCT

382 kb

upstream

upstream of Plg GCAGAAAGAGGGGATATAGTCAGTG AGGGGTTACAACTTGTCGAATGAC

363 kb

upstream

near Plg promoter CTCCCTGTTCGAGGTAATGT CCTAGTTCCTGGTGTTGACTTA

342 kb

upstream

Plg intron 10 GTGTATAACACCTCCACTCCAAC CTGCACTGAAGTCTATCTAACAGC

325 kb

upstream

Plg intron 17 GTATCTCCACTCCTGGACTTAGTT CTGTCTGGTGATTATAGGAGCTG

285 kb

upstream

Slc22a3 intron 5 GAGCTGCATACTGGTTAAGAACG CCCCAAAGAGTAACAGGATAGAAG

234 kb

upstream

Slc22a3 promoter

(-57 ~ +38)

CAGGTATGAAACTCTGTTCCTG GGAATAGCCTCCAGTACTCTTAG

234 kb

upstream***

Slc22a3 promoter

(-319 ~ -223)

GCTCTGTAGATTTTTGAGGAGCAAG ATATCCCCGCATCCAGACTCTC

234 kb

upstream***

Slc22a3 promoter

(-597 ~ -498)

GGTTTCTCAGCATCCCGAGTAGTA AGGGCTCAGATTTAGTCCCCTTC

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224 kb

upstream

between Slc22a3

and Slc22a2

GTGTGGTTATTGACAGTGTGAC GACAGTTTCTCATCATGACTCC

204 kb

upstream

between Slc22a3

and Slc22a2

AGACAGATCTTACCCATGTCCA GCTGTATACCTGCTCTGCTCTC

157 kb

upstream***

Slc22a2 promoter

(-581 ~ -492)

TTTCCTCACAAGTGGGTGAAGAG CCTTCTGACTTTTCAAAGGGTTCAT

157 kb

upstream***

Slc22a2 promoter

(-338 ~ -229)

GACAGGAGAGGCCAGCTGTATAGG GAGAAATCTGAGTCTCTTGGCATA

TCA

157 kb

upstream

Slc22a2 promoter

(-190 ~ -56)

TGTGAAGCAGAGACGAACTCACAG ACTTCCCTCAGACAGGCCAACTT

142 kb

upstream

Slc22a2 exon 3 GTCACCATCCTCATCAATGCTATC ACCAGTCCCTGTAGAAATCGAAAC

103 kb

upstream

between Slc22a2

and Slc22a1

GCAGATACCAGATCTACAAAGC AAGACACTGGCAGACCTAACT

76 kb

upstream

Slc22a1 intron 3 CTAACTCAGCCTTACTCCTCTG GCTTAGCCTCACAGATACAGTAG

66 kb

upstream

near Slc22a1

promoter

CTTTAGCTCCCATCTACGTG GGTGTAGTTCAGCTCCTCTG

51 kb

upstream

Igf2r intron 41 TGGATGCTAGTCATATAGGTGTGG AGTTACTGGCCCATACACTCAAC

16 kb

upstream

Igf 2r intron 9 CTTAAGCTGCTAGTTGGCTTCACA CGTCCAAAACCACCATCTACAAG

0.1 kb

upstream

Air ptomoter CTGTAAGGCCATCTAAAACACAGG GCAGGGGGTGGAGATTATTAAGT

2 kb near 5' end of Air TGGACCGAGTGATAAGAACTAC GGCTATTGCTAAGTGGCTACTAC

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downstream

28 kb

downstream***

Igf2r intron 1 ACCAACAAACTTCGACAGGACT CTTTACCCTTCACGTCTCTCAGTT

29 kb

downstream

Igf2r promoter AGACTGACCTCTTAACCCTGCAT CTCTTCTTCAACCGAGACCAGTA

65 kb

downstream

middle of Air ACTTTGACAGAACAATCGGCTCAG GAACATTTGCAAAGGACAGTCGAG

85 kb

downstream

Au76 pseudogene ATTCTGTGGCATACGACCTCTTC GTGGCTGACCAAAATCTCCTG

106 kb

downstream**

3' end of Air CTACAGGTTGTCACTACTTCCAC GACTGCTCTAGCAGGAAGTATC

121 kb

downstream**

Mas1 promoter GCTACAGCTATCTTCCTAAACCAG AGGTCTGGTGAGAACACTCTATG

~ 2.3 Mb

downstream

Chd1 intron 1 GCTAAGTCCTAGCAAGCAAGAATG ATGTAAGCCCGACTTAGAACTCC

*Based on Ensembl mouse Release 41 unless stated otherwise.

**Based on GenBank AJ249895 (different from Ensembl).

***These primers were used only in ChIP analyses for histone modification.

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Supporting references

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