Hindiii enzyme

4C-seq assays were performed according to Splinter et al [72 (link)] with slight modifications. Briefly, 4 × 10⁷ cells were crosslinked with 1% formaldehyde. The nuclei pellets were isolated by cell lysis with cold lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 5 mM EDTA, 0.5% NP 40) supplemented with protease inhibitors (Roche). First step digestion was performed overnight at 37 °C with HindIII enzyme (NEB). Digestion efficiency was measured by RT-qPCR with HindIII site-specific primers. After confirmation of good digestion efficiency, DNA was ligated overnight at 16 °C by T4 DNA ligase (Thermo Scientific) and de-crosslinked. Following de-crosslinking, DNA was extracted by phenol-chloroform and this is the 3C library. The DNA was then processed for second digestion with DpnII enzyme (NEB) overnight at 37 °C. After final ligation, 4C template DNA was obtained, and the concentration was determined using Qubit assays (Thermo Scientific). The 4C template DNA was then amplified using specific primers with Illumina Nextera adapters and sent for sequencing on the MiSeq system. All the 4C genome coordinates are listed in Additional file 1: Table S9.

4 C was performed as previously described^{58 (link),59 (link)}. Cells were fixed with 2% formaldehyde for 10 min, cross-linked chromatin was digested overnight with an excess of HindIII enzyme (New England Biolabs) and then DNA ends were ligated under dilute conditions that favor junctions between cross-linked DNA fragments. The ligation junctions were then circularized by digestion with Csp6I (Thermo Scientific). Chromosomal contacts with the baits were amplified with inverse PCR primers (Supplementary Table S2) using Platinum Taq DNA Polymerase (LifeTechnologies). 4C-seq libraries were sequenced on Illumina Hi-seq 2000 platform.

Production of the short gRNA (100 nt in length) specific to dpy-10 gene was performed according to the protocol described in (Hwang et al. 2013 (link)). In brief, a plasmid encoding gRNA (targeting dpy-10) was constructed as follows: pDR274 vector for in vitro gRNA production (a gift from Keith Joung, Addgene plasmid # 42250; https://www.addgene.org/42250/; RRID: Addgene 42250) containing a T7 promoter upstream of gRNA scaffold sequence was digested with BsaI enzyme (NEB, Cat. No. R3733S). It was then used as a backbone for cloning the annealed oligonucleotides (dpy-10T: 5′-TAGGGCTACCATAGGCACCACGAG-3′; dpy-10B: 5′-AAACCTCGTGGTGCCTATGGTAGC-3′), containing dpy-10 protospacer sequence (5′-GCTCGTGGTGCCTATGGTAG-3′). The sequence verified expression vector was then digested with HindIII enzyme (NEB, Cat. No. R3104S) and used as a template for in vitro transcription of gRNA by AmpliScribe T7 High Yield Transcription Kit (Epicentre Technologies, Cat. No. AS3107).

To ensure the isolation of DNA of the expected size, two partial restriction digests were performed to determine the conditions yielding an appropriate percentage of fragments between 100 and 350 kb in size. Size selection was performed according to previously described protocols using the HindIII enzyme (New England Biolabs, UK) [19 (link), 21 ]. The optimal conditions were determined by digesting the chopped plugs at several enzyme concentrations (0.2–50 U). The macerated plug pieces were placed in 1.5-mL Eppendorf tubes containing the appropriate buffer and the HindIII enzyme and incubated on ice for 1 hour. Digestion was performed at 37°C for 10 min, and the reaction was stopped by adding 30 μL of 0.5 M EDTA (pH 8.0).
Partially digested HMW DNA was separated via PFGE (pulsed-field gel electrophoresis) in 1% TBE agarose (CHEF-DR II drive module, Bio-Rad) using the following parameters: 120° angle, 12°C, 6.0 V/cm, initial switch time = 1.0 sec, final switch time = 40.0 sec, ramping = linear, and running time = 18 hours. Once the optimal HindIII concentration was determined, mass digestion was performed using 13 plugs. The digested HMW DNA was separated into two selected sizes via PFGE, as described by Peterson et al. (2000) [21 ].
The DNA inserts embedded in agarose gel pieces were maintained in 50-mL polypropylene centrifuge tubes containing 1× TAE at 4°C before electroelution.

Interactions between enhancer and nearby gene promoter were determined by 3C and quantified by quantitative real-time PCR (qPCR). In summary, ~ 10⁸ HepG2 cells were cross-linked by formaldehyde and lysed, and the chromatin was digested by HindIII enzyme (NEB). After ligation with T4 DNA ligase (NEB), DNA was purified.
Along with the chromatin, the BAC RP11-121J8 was ordered from BACPAC Resources Center (http://bacpac.chori.org/), cultured, isolated by Large-Construct Kit (Qiagen, Valencia, CA), digested with the same enzyme and ligated as control.
The relative amount of 3C product was quantified by qPCR with iQ SYBR green (Bio-Rad, Hercules, CA) and unidirectional primers in Supplementary Table 9. The relative enrichment for HepG2 chromatin was determined by 2^-ΔΔCt, in which ΔΔCt is the threshold cycle difference between BAC and chromatin. Triplicates were performed for each 3C-qPCR. All 3C products were sequenced for validation.

3C was utilized to decipher the spatial genome organization between distal enhancer and promoter as previously reported [11 (link)]. In brief, formaldehyde (1% final concentration) was utilized to crosslink Beas-2B or A549 cells (approximately 10⁸). After lysing by Lysis Buffer, the crosslinked chromatin DNA was digested by HindIII enzyme (NEB) and ligated by T4 DNA ligase (NEB). The 3C library was extracted by phenol-chloroform method and DNA quantification was performed in Qubit^® 3.0 fluorometer (Thermo Fisher Scientific).
Meanwhile, bacterial artificial chromosome (BAC) containing partial 15q15.2 region, RP11-1012I24 (BACPAC Genomics, Richmond, CA), was cultured for 16 h with 280 rpm shaking at 37°C and extracted by Large-Construct Kit (Qiagen, Valencia, CA) following the manufacturer’s introduction. After digestion by HindIII, the BAC was ligated and purified by the abovementioned method.
The relative enrichment of 3C product was assessed by quantitative PCR (qPCR) with iQ SYBR green (Bio-Rad, Hercules, CA) and primers in S3 Table. The formula 2^–ΔΔCt was utilized to quantify the relative enrichment for chromatin. Three replicates were executed for each primer pair. The qPCR products were verified by resequencing.