Supplementary data
I. Supplementary Figs
Supplementary Fig1 Confirming the digestion efficiency of cross-linked DNA in 3C assay by
Agarose electrophoresis, southernblotting, semi-quantitative PCR. In the electrophoresis experiment,
freshly prepared mouse genome DNA was the control. CL DNA/NcoI represent the DNA extracted
from the cross linked and NcoI digested nuclei of mouse fetal liver cells. The following probes were
used in southernblotting: HS8, a 502-bp PCR fragment, which hybridizes to a 2.2-kb NcoI HS8
fragment, a 485-bp PCR fragment, which hybridizes to a 2.2-kb NcoI  fragment; 1, a 388-bp
PCR fragment, which hybridizes to a 2.2-kb NcoI 1 fragment; 2, a 420-bp PCR fragment, which
hybridizes to a 1.5-kb NcoI 2 fragment. The primers for amplifying the probes are as follows: HS8L, GATCTACAGACTGCCCTCCCAAGTC; HS8-R, TATAAAGTGCTTTCCCTCACCAGGG; -L,
CATAGCCATTTGTTGCCAATCAGTG;
-R,
GGGCTTCATAGTGAGACCGCATC;
1-L,
TGCTCACATCCATTCAGACACAGAC; 1-R, AGGTTGGGACAAGTACAGTTAGGG; 2-L,
GCTGCCCTTCCCTCATCCTCTG; 2-R, AAATCCGGTTG TTACTTGATCATGC. In the semiquantitative PCR assay, the primers were designed at the two sides of NcoI sites. The mouse genome
DNA is the positive control and the mouse genome completely digested by NcoI is the negative
control.
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Supplementary Fig2 Position of 475A8 on mouse chromosome 11. The position of captured
475A8 RCF belongs to BAC clone RP23-475A8. The whole BAC clone includes more than 200kb
DNA sequence. There is one functional gene Fam44B in the whole BAC clone which is essential for
the mitotic spindles assembly. The numbers corresponding to the sequence number of NCBI
Mapviewer number. The 475A8 RCF is nearly 1.7kb and the Vista program identified two closely
located highly conserved regions, CR1 and CR2, were located at the position from 0.7kb to 1.3kb of
this RCF.
Supplementary Fig3 Confirmation of GATA-1 expression of GATA-1 expression vector in
GP293 cells. GP293 is a cell line that has no GATA-1 expression. RT-PCR was performed to detect
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the GATA-1 expression after the GP293 cells being transfected by GATA-1 expression vector. The
total RNAs were the negative control.
Supplementary Fig4 Increased -globin gene expression in MEL cells induced by DMSO. RTPCR was performed to confirm that -globin gene expression increased accompany with the DMSO
BAC NAME
RP24-163H9
RP24-367P22
RP23-107I8
RP23-188H3
RP23203H20
RP23398G11
RP23386K17
TAG
POSITION
-75675
113380
22729
6935
55049
CHROMOSOME
TAG SEQUENCE
15
15
10
11
10
CCATGGTTCAAAGGAAGAGAAA
CCATGGACTCTCCATTTATCT
CCATGGCTTTCTGTTATATG
CCATGGCACCCAAGAGGTCCT
CCATGGCTTTGTCACCAGGTT
3C
SIGNAL/42CYCLES
yes
yes
yes
yes
yes
148314
11
CCATGGCTTTGCTTTGCTTGGATTC
yes
-52307
11
CCATGGTTTTTGCATCCGCTCCTGCCTT
yes
induction of MEL cells for 1 day to 4 days. GAPDH was the control.
3
RP23-12D16
RP24175C20
RP23-30H7
113165
-136021
8
9
211765
14
CCATGGAGGCAGATCACATGCT
CCATGGAAAGGTGTTTTTGC
yes
no
CCATGGAGCCATCCGTTCTCT
no
II Supplementary Table
Supple table1 3C verification of the low frequency tags. The 3C assay of 10 randomly selected low
frequency tags. The PCR is performed as following: one cycle at 94°C for 4 min; 42 cycles at 94°C
for 30 s, 58°C for 40 s and 72°C for 30 s; followed by one cycle at 72°C for 10 min. Position number
represents the tag position in the BAC sequence. Tag sequences are all beginning with NcoI
recognition sequences that represent the original NcoI digestion site during 3C template preparation.
III. Primers used in 3C assay
Primer
sequences
for
the
tested
restriction
fragments
were
as
follows:
HS26,
GTATGGTAGCTTACCCCTATAATGCC ;475A8, AGGAGAAAGGTCACCCATGTCA ; MPG,
CACAGAA
-globin,
-globin, GACAGTGCAGGTCTGGATACAAGA. Errc3-1
(CTATATTCAACTGCTGTTCCCATG) and Errc3-2 (TTCTACCAGCAGATC CGTATTCC)
IV. Step by step description of QACT
Step 1, Crosslink the cells.
Purpose, fix the in vivo chromatin status.
Step2, NcoI, the first enzyme digestion and religation
Purpose, generating the cohesive ends by NcoI that could religate to form the hybrid molecules
(spatially proximal DNA will join together)
Step3, Reverse Crosslink
Purpose, to purify the hybrid molecules for next step including either 3C PCR or QACT
Step4, Digest the hybrid molecules by TfiI, the secondary enzyme and self-ligation
Purpose, To make the hybrid molecules more small and facilitate the intramolecular ligation(TfiI end
joining).
The circular molecule will act as the template of inverse PCR.
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Step5, Inverse PCR
Purpose, Using the primers that locate at the HS26 fragment(Known part of the hybrid fragment) to
amplify the ACPs(unknown part of the hybrid fragment)
Step6, Replace the HS26 fragment at one side of the PCR product by NcoI adapor
Purpose, NcoI adapor includes MmeI recognition site and MmeI can excise 19/20bp ACPs through
recognize its binding site at the NcoI adapor.
Step7, MmeI digestion
Purpose, Remove unnecessary sequence except 19/20 bp short tags that are together with NcoI adaptor.
Step8, Adding NN adaptor to the cohesive end generated by MmeI digestion
Purpose, Facilitate the amplification of the tags.
Step9, Amplification of the tags by PCR and excise the 19/20bp fragment by BamHI and PstI
digestion.
Purpose, amplifying and releasing the tags from the adapor-tag-adapor structure and facilitate the self
ligation of these tags (concatemers)
Step10, Self ligation of the tags to concatemers and sequencing.
Purpose, it is more efficient to sequence the concatemers to get the sequence information of tags.
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