The long-range interaction landscape of gene promoters methods
Aim. Evidence-backed execution summary for The long-range interaction landscape of gene promoters methods from The long-range interaction landscape of gene promoters.
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What do I need before I start?
human
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Supplemental Methods
reagent used in the protocol.
- Use
- 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...
Formaldehyde crosslinking
reagent used in the protocol.
- Use
- 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 formalde...
5C analysis
reagent used in the protocol.
- Use
- 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 overlappi...
Generation of 5C libraries
reagent used in the protocol.
- Use
- 3C was performed with Hin dIII restriction enzyme as previously described, for GM12878, K562 and HeLa-S3 cells separately with two biological replicates for each cell line. The 3C libraries were then interrogated by 5C. The 44 ENCODE regions were analyzed in two groups using two separate 5C primer pools. The first...
Generation of 5C libraries
reagent used in the protocol.
- Use
- The primer pool for the ENm group contained a total of 3,150 primers (476 reverse 5C primers and 2674 forward 5C primers). This primer pool allows interrogation of a total of 1,272,824 interactions. Of these, 83,427 interactions were between fragments that were both located in the same ENCODE region. The primer pool...
Generation of 5C libraries
reagent used in the protocol.
- Use
- 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...
Generation of 5C libraries
reagent used in the protocol.
- Use
- To facilitate Illumina paired end DNA sequence analysis of 5C libraries, Illumina paired end adapter oligos (Illumina, San Diego, CA) were ligated to the 5C library using the Illumina PE protocol. The linkered 5C library was then amplified by PCR (17 or 18 cycles, with Phusion High Fidelity DNA polymerase) using Ill...
5C read mapping
reagent used in the protocol.
- Use
- The fastQ files were taken directly from the Illumina GAIIx and fed into our in-house 5C mapping pipeline. Each side of the paired end read was independently mapped to a pseudo-genome of all possible 5C primer sequences using the novoalign mapping algorithm (V2.05 http://novocraft.com ). The default alignment settin...
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 overlappi...
- Use
- 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 overlappi...
Generation of 5C libraries
To facilitate Illumina paired end DNA sequence analysis of 5C libraries, Illumina paired end adapter oligos (Illumina, San Diego, CA) were ligated to the 5C library using the Illumina PE protocol. The linkered 5C library was then amplified by PCR (17 or 18 cycles, with Phusion High Fidelity DNA polymerase) using Ill...
- Use
- To facilitate Illumina paired end DNA sequence analysis of 5C libraries, Illumina paired end adapter oligos (Illumina, San Diego, CA) were ligated to the 5C library using the Illumina PE protocol. The linkered 5C library was then amplified by PCR (17 or 18 cycles, with Phusion High Fidelity DNA polymerase) using Ill...
5C read mapping
Sequencing data was obtained from an Illumina GAIIx machine and was processed through a custom pipeline to map and assemble 5C interactions. We used thirty six (36) base-pair paired end reads to sequence all 5C libraries. Due to sequencing efficiency some 5C libraries were re-sequenced as many as 10 times to obtain...
- Use
- Sequencing data was obtained from an Illumina GAIIx machine and was processed through a custom pipeline to map and assemble 5C interactions. We used thirty six (36) base-pair paired end reads to sequence all 5C libraries. Due to sequencing efficiency some 5C libraries were re-sequenced as many as 10 times to obtain...
5C read mapping
The fastQ files were taken directly from the Illumina GAIIx and fed into our in-house 5C mapping pipeline. Each side of the paired end read was independently mapped to a pseudo-genome of all possible 5C primer sequences using the novoalign mapping algorithm (V2.05 http://novocraft.com ). The default alignment settin...
- Use
- The fastQ files were taken directly from the Illumina GAIIx and fed into our in-house 5C mapping pipeline. Each side of the paired end read was independently mapped to a pseudo-genome of all possible 5C primer sequences using the novoalign mapping algorithm (V2.05 http://novocraft.com ). The default alignment settin...
5C read mapping
Statistics regarding the 5C library quality, mapping efficiency etc. can be found in. Since it is only necessary to map the paired end reads to the list of all possible 5C primers rather than to the entire genome, a higher percentage of mapped/usable reads can be achieved. We find that > 90% of all paired reads (af...
- Use
- Statistics regarding the 5C library quality, mapping efficiency etc. can be found in. Since it is only necessary to map the paired end reads to the list of all possible 5C primers rather than to the entire genome, a higher percentage of mapped/usable reads can be achieved. We find that > 90% of all paired reads (af...
Detection bias correction
5C experiments involve a number of steps that can locally differ in efficiency, thereby introducing biases in efficiency of detection of pairs of interactions. These biases could affect the efficiency of cross-linking, the efficiency of restriction digestion (related to cross-linking efficiency), the efficiency of l...
- Use
- 5C experiments involve a number of steps that can locally differ in efficiency, thereby introducing biases in efficiency of detection of pairs of interactions. These biases could affect the efficiency of cross-linking, the efficiency of restriction digestion (related to cross-linking efficiency), the efficiency of l...
Before you run
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First confirmation
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Open the source paper before finalizing run-specific details.
Procurement checkpoint
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Open quote workflowStep-by-step procedure
What do I do, in order?
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...
Measurement outputs
What raw and processed outputs should exist?
5C analysis was carried out as previously described, for the 44 ENCODE Pilot regions (ENCODE Manual - ENm and ENCODE Random - Enr). The chromosomal position and coo...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
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.umassme...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
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 react...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
To facilitate Illumina paired end DNA sequence analysis of 5C libraries, Illumina paired end adapter oligos (Illumina, San Diego, CA) were ligated to the 5C library using the Il...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Statistics regarding the 5C library quality, mapping efficiency etc.
from paperScoring or quantification
Quantify the primary readouts for this experiment: 5C analysis was carried out as previously described, for the 44 ENCODE Pilot regions (ENCODE Manual - ENm and ENCODE Random - Enr). The chromosomal position and coo...; 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.umassme...; 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 react...; To facilitate Illumina paired end DNA sequence analysis of 5C libraries, Illumina paired end adapter oligos (Illumina, San Diego, CA) were ligated to the 5C library using the Il....
from paperStatistical comparison
Statistics regarding the 5C library quality, mapping efficiency etc. can be found in. Since it is only necessary to map the paired end reads to the list of all possible 5C prim...; To detect significant looping interactions from background looping interactions we developed an in-house "5C peak calling" algorithm. We chose to call peaks in each...; 5C signals represent the three-dimensional contact probabilities between pairs of loci. This relationship inversely scaled with genomic distance. To properly control for the var...; We define a false-positive 5C looping interaction as an interaction that is identified in the peak calling approach described above but is due to technical biases or noise and t...
from paperReporting output
Report representative outputs alongside summary comparisons for 5C analysis was carried out as previously described, for the 44 ENCODE Pilot regions (ENCODE Manual - ENm and ENCODE Random - Enr). The chromosomal position and coo..., 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.umassme..., 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 react..., To facilitate Illumina paired end DNA sequence analysis of 5C libraries, Illumina paired end adapter oligos (Illumina, San Diego, CA) were ligated to the 5C library using the Il....
inferred from protocolStructured statistical methods
Statistics regarding the 5C library quality, mapping efficiency etc. can be found in. Since it is only necessary to map the paired end reads to the list of all possible 5C prim...; To detect significant looping interactions from background looping interactions we developed an in-house "5C peak calling" algorithm. We chose to call peaks in each...; 5C signals represent the three-dimensional contact probabilities between pairs of loci. This relationship inversely scaled with genomic distance. To properly control for the var...; We define a false-positive 5C looping interaction as an interaction that is identified in the peak calling approach described above but is due to technical biases or noise and t...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
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...
Machine-readable layer
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