The long-range interaction landscape of gene promoters methods
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Supplemental Methods
GM12878 lymphoblastoid cells were procured from Coriell Cell Repositories and grown in RPMI 1640 medium supplemented with 2mM L-glutamine, 15% fetal bovine serum (FBS) and antibiotic (1% Pen-Strep). K562 (CCL-243), a CML cell line and HeLa-S3 (CCL2.2), a cervical carcinoma cell line were obtained from American Type Culture Collection (ATCC). K562 cells were cultured in similar media as GM12878 except with 10% FBS while HeLa-S3 cells were maintained in ATCC recommended F-12K Medium (Kaighn's Modification of Ham's F-12 Medium) with 10% FBS and 1% Pen-Strep. The culture densities and conditions were maintained as per recommendations of the repositories.
Formaldehyde crosslinking
For suspension cells (GM12878, K562) cells a total of 1 × 10ˆ8 freshly growing cells were centrifuged at 100Xg for 5 minutes. Cell pellets were resuspended in 45 mL of respective growth medium in a 50 mL Falcon tube. Cells were fixed by addition of 1.25 mL of 37% formaldehyde (final concentration of formaldehyde 1%). The cell suspension was gently mixed by inverting the tube up and down 4-6 times at room temperature and the tubes was rotated on an end-to-end shaker for exactly 10 minutes. Crosslinking was stopped by addition of 3M glycine (final concentration 125 mM) and cell suspensions were incubated at room temp for 15 minutes using an end-to-end shaker. The crosslinked cells were then pelleted at 100Xg for 5 minutes and the cell pellet was stored at -80°C. For HeLa-S3, the adherent cells were first trypsinized and then the crosslinking was performed as described above.
5C analysis
5C primers were designed at Hin dIII restriction sites (AAGCTT) using 5C primer design tools previously developed and made available online at My5C website ( http://my5C.umassmed.edu ). Reverse 5C primers were designed for Hin dIII restriction fragments overlapping a known TSS from GENCODE transcripts, or overlapping a start site as experimentally determined by CAGE Tag data of the ENCODE pilot project ( ). Forward 5C primers were designed for remaining of the Hin dIII restriction fragments ( ). For ENCODE regions that do not contain any TSS according to gene annotation in 2008 (ENr112, ENr113, ENr311 and ENr313) we employed an alternative primer design. For these regions an alternating design of forward and reverse 5C primers was used in which forward and reverse primers are designed for alternating restriction fragments. Note that ENr311 contains genes according to 2011 GencodeV7...
Generation of 5C libraries
5C was performed in 10-15 reactions each containing an amount of 3C library that represents 200,000 genome equivalents and 0.5 fmol of each primer. The multiplex annealing reaction was performed overnight at 55 °C. Pairs of annealed 5C primers were ligated at the same temperature using Taq DNA ligase for 1 hour. Ligated 5C primer pairs, which represent a specific ligation junction in the 3C library and thus a long-range interaction between the two corresponding loci, were then amplified using 28 cycles of PCR with universal tail primers that recognize the common tails of the 5C forward and reverse primers. At least four separate amplification reactions were carried out for each 10-15 annealing reactions described above and all the PCR products were pooled together. This pool constitutes the 5C library. The libraries were concentrated using Qiaquick PCR purification kit and 3̸...
Estimation of frequency of false positive looping interactions
The false positive detection rate for single replicates can be calculated in exactly the same way. We find that the fraction of significant looping interactions detected in one replicate that might be false positives ranges from 20% to 47%. Thus, by requiring interactions to be significant in both replicates we greatly reduce the fraction of false-positive significant interactions (from 20%-47% to 10-18% of the significant interactions). At the same time, many of significant interactions detected in only one replicate are not false-positives, and by excluding this subset of interactions from our analysis we introduce false-negatives. Consistent with our interpretation that many of the peaks seen in only one replicate represent false-negatives, we find that when we take the union of the peaks found in replicate 1 and 2, or analyze the set of peaks obtained with individual replicates se...
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GM12878 lymphoblastoid cells were procured from Coriell Cell Repositories and grown in RPMI 1640 medium supplemented with 2mM L-glutamine, 15% fetal bovine serum (FBS) and antibiotic (1% Pen-Strep). K562 (CCL-243), a CML cell line and HeLa-S3 (CCL2.2), a cervical carcinoma cell line were obtained from American Type Culture Collection (ATCC). K562 cells were cultured in similar media as GM12878 except with 10% FBS while HeLa-S3 cells were maintained in ATCC recommended F-12K Medium (Kaighn's Modification of Ham's F-12 Medium) with 10% FBS and 1% Pen-Strep. The culture densities and conditions were maintained as per recommendations of the repositories.
For suspension cells (GM12878, K562) cells a total of 1 × 10ˆ8 freshly growing cells were centrifuged at 100Xg for 5 minutes. Cell pellets were resuspended in 45 mL of respective growth medium in a 50 mL Falcon tube. Cells were fixed by addition of 1.25 mL of 37% formaldehyde (final concentration of formaldehyde 1%). The cell suspension was gently mixed by inverting the tube up and down 4-6 times at room temperature and the tubes was rotated on an end-to-end shaker for exactly 10 minutes. Crosslinking was stopped by addition of 3M glycine (final concentration 125 mM) and cell suspensions were incubated at room temp for 15 minutes using an end-to-end shaker. The crosslinked cells were then pelleted at 100Xg for 5 minutes and the cell pellet was stored at -80°C. For HeLa-S3, the adherent cells were first trypsinized and then the crosslinking was performed as described above.
5C primers were designed at Hin dIII restriction sites (AAGCTT) using 5C primer design tools previously developed and made available online at My5C website ( http://my5C.umassmed.edu ). Reverse 5C primers were designed for Hin dIII restriction fragments overlapping a known TSS from GENCODE transcripts, or overlapping a start site as experimentally determined by CAGE Tag data of the ENCODE pilot project ( ). Forward 5C primers were designed for remaining of the Hin dIII restriction fragments ( ). For ENCODE regions that do not contain any TSS according to gene annotation in 2008 (ENr112, ENr113, ENr311 and ENr313) we employed an alternative primer design. For these regions an alternating design of forward and reverse 5C primers was used in which forward and reverse primers are designed for alternating restriction fragments. Note that ENr311 contains genes according to 2011 GencodeV7 annotation. Primers were excluded for highly repetitive sequences that prevented the design of a sufficiently unique 5C primer. Primers settings were as described before: U-BLAST: 3; S-BLAST: 130: 15-MER: 1320; MIN_FSIZE: 40; MAX_FSIZE: 50000; OPT_TM: 65; OPT_PSIZE: 40. The 5C primers contained up...
5C was performed in 10-15 reactions each containing an amount of 3C library that represents 200,000 genome equivalents and 0.5 fmol of each primer. The multiplex annealing reaction was performed overnight at 55 °C. Pairs of annealed 5C primers were ligated at the same temperature using Taq DNA ligase for 1 hour. Ligated 5C primer pairs, which represent a specific ligation junction in the 3C library and thus a long-range interaction between the two corresponding loci, were then amplified using 28 cycles of PCR with universal tail primers that recognize the common tails of the 5C forward and reverse primers. At least four separate amplification reactions were carried out for each 10-15 annealing reactions described above and all the PCR products were pooled together. This pool constitutes the 5C library. The libraries were concentrated using Qiaquick PCR purification kit and 3′-A tailing reaction was done using dATP and Taq DNA polymerase in presence of 1X standard Taq buffer (NEB) at 72°C for 30 minutes.
The false positive detection rate for single replicates can be calculated in exactly the same way. We find that the fraction of significant looping interactions detected in one replicate that might be false positives ranges from 20% to 47%. Thus, by requiring interactions to be significant in both replicates we greatly reduce the fraction of false-positive significant interactions (from 20%-47% to 10-18% of the significant interactions). At the same time, many of significant interactions detected in only one replicate are not false-positives, and by excluding this subset of interactions from our analysis we introduce false-negatives. Consistent with our interpretation that many of the peaks seen in only one replicate represent false-negatives, we find that when we take the union of the peaks found in replicate 1 and 2, or analyze the set of peaks obtained with individual replicates separately all the results we presented remain the same: 1) enrichment for distal elements that resemble active gene regulatory elements ( ); 2) Asymmetry of the long-range interaction landscape with a peak around 120 Kb upstream of the TSS ( ); 3) Skipping over CTCF sites and 4) Formation of interwov...
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