Inverse PCR

The 3C-seq experimental procedure is outlined in Figure 1, and the subsequent r3Cseq data analysis workflow is shown in Figure 2. Isolated cells are treated with a cross-linking agent to preserve in vivo nuclear proximity between DNA sequences. The DNA isolated from these cells is then digested using a primary restriction enzyme, typically a 6-bp cutting enzyme, such as HindIII, EcoRI or BamHI. The digested products are then ligated under diluted conditions to favor intra-molecular over inter-molecular ligation events. This digested and ligated chromatin yields composite sequences representing (distal) genomic regions that are in close physical proximity in the nuclear space. The digested and ligated chromatin is then de-cross-linked and subjected to a second restriction digest using a four-cutter (e.g. NlaIII or DpnII) as a secondary restriction enzyme to decrease the fragment sizes. The resulting digested DNA is then ligated again under diluted conditions, creating small circular fragments. These fragments are inverse PCR amplified using primers specific for a genomic region of interest (e.g. promoter, enhancer or any other element potentially involved in long-range interactions), termed the ‘viewpoint’. The amplified fragments are then sequenced using massively parallel high-throughput sequencing. The 3C-seq procedure produces DNA molecules consisting of viewpoint-specific primers followed by sequences derived from the ligated interacting fragments. These need to be trimmed in silico to remove the primer and viewpoint sequence, thus leaving only the captured sequence fragments for mapping (14 (link)). After trimming, reads are mapped against a reference genome using alignment software, such as Bowtie (18 (link)).
Figure 1.

3C-seq experimental procedures and data analysis workflow. Formaldehyde cross-linked chromatin is digested with a six-cutter restriction enzyme and ligated under dilute conditions. After de-cross-linking, DNA is digested with a four-cutter enzyme and again ligated under dilute conditions to create small circular fragments representing individual ligation events. Inverse PCR using viewpoint-specific primers containing Illumina sequencing adapters is used to generate a viewpoint-specific 3C-seq library. After high-throughput sequencing, reads are trimmed and mapped to the reference genome, after which they are loaded into the r3Cseq software.

Figure 2.

A summary of the r3Cseq analysis pipeline. The main features and the sequential order of operations are shown in the flow chart. In-depth discussion of the different operations and functions can be found in the ‘Materials and Methods’ and ‘Results’ sections.

Our r3Cseq package has been developed in the R statistical framework (19 ) as part of Bioconductor (20 (link)). It uses binary alignment/map (BAM)-aligned read files as input (21 (link)), which are generated by commonly used alignment software and carries out operations, such as class initialization, counting aligned reads per restriction fragment or per window size, read count normalization, statistical analysis of interactions in both cis and trans, data visualization and data export of the identified contacting regions. Figure 2 shows the main features and the sequential steps of the r3Cseq pipeline.

All-in-one doxycycline-inducible lentiviral transfer plasmids (pLV-Dox mCherry-FP4/AP4-MITO) were generated by PCR and subcloning mCherry-FP4-MITO or mCherry-AP4-MITO (a gift from James Bear, University of North Carolina, Chapel Hill, NC, USA; Bear et al., 2000 (link)) in place of Cas9 in pCW-Cas9 (a gift from Eric Lander and David Sabatini (Massachusetts Institute of Technology, Cambridge, MA, USA; Wang et al., 2014 (link); RRID:Addgene_50661). Briefly, Cas9 was cut out and replaced with a multiple cloning site by annealed oligo cloning, and AgeI-BamHI was used to insert FP4/AP4 sequences. TurboRFP-expressing pLKO transfer plasmid was generated by PCR and subcloning TurboRFP in place of the puromycin resistance gene at BamHI-KpnI in pLKO.1 - TRC cloning vector (a gift from David Root, The Broad Institute, Cambridge, MA, USA; Moffat et al., 2006 (link)]; RRID:Addgene_10878). Validated Mus musculus shRNA sequences were obtained from The RNAi Consortium (The Broad Institute via MilliporeSigma; Moffat et al., 2006 (link)) and oligos were ligated between the AgeI-EcoRI sites (replacing the 1.9 kb stuffer) of our pLKO.1 TurboRFP cloning vector using annealed oligo cloning. shRNA sequences and source MilliporeSigma product number are given in Table S1. Lentiviral LifeAct expression vectors were published previously (Padilla-Rodriguez et al., 2018 (link); Parker et al., 2018 (link)): pLenti LifeAct-EGFP BlastR (RRID:Addgene_84383), pLenti-LifeAct-mRuby2 BlastR (RRID:Addgene_84384), pLenti LifeAct-iRFP670 BlastR (RRID:Addgene_84385). Lentiviral cDNA expression vectors were generated by PCR and subcloning cDNA of interest into the transfer plasmids pLenti CMVie-IRES-BlastR or pLenti CMVie-IRES-BlastR alt MCS (pCIB) published previously (Puleo et al., 2019 (link); RRID:Addgene_119863 and RRID:Addgene_120862). To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; Lam et al., 2012 (link); RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; Shcherbakova and Verkhusha, 2013 (link); RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC. To generate lentiviral expression vectors for PSD95-FingR-EGFP and -mRuby2, pCAGGs-PSD95-FingR-EGFP was cut with SpeI-AgeI and ligated into pCIB, and mRuby2 was subcloned into SmaI-AgeI sites. For EVL iLID and MIM iLID optogenetic systems, tgRFPt-SspB(R73Q) was PCRed and subcloned from pLL7.0 mTiam1(64–437)-tgRFPt-SSPB R73Q (a gift from Brian Kuhlman, University of North Carolina, Chapel Hill, NC, USA; Guntas et al., 2015 (link); RRID:Addgene_60418) and added to the N-terminus of EVL or C-terminus of MTSS1. Lentiviral expression vectors for MIM-mRuby2, MIM-iRFP670, and MIM iLID were modified to remove the IRES-blasticidin resistance cassette to reduce plasmid size. Additional mammalian expression vectors for co-immunoprecipitation experiments were subcloned into pEF1-MCS-mychis6 B (V92120; Invitrogen) or pCMV 3xFLAG-MCS (Parker et al., 2013 (link)). Mutants of EVL and MIM were generated by PCR or inverse PCR and self-ligation cloning. All plasmids created for this paper will be made available through Addgene where possible. Complete list of plasmids generated and used in this paper can be found in Table S1.