GSM1569155: PE-4CSeq from MEF line 129S5E117, viewpoint 155.5 (12), allele 129S5; Mus musculus; OTHER
4C templates for Df/+Bl6 (MEF lines 129S5E71 and 129S5E98) and +129/+Bl6 (MEF lines 129S5E117 and 129S5E118) were prepared as described in Splinter et al, 2012. Briefly, 1x107 MEFs were cross-linked for 10 min at RT using 2% formaldehyde (Calbiochem) and 10%FCS in PBS (pH 7.4). 10 ml reactions were transferred to ice and added 1.425ml of 1M glycine, followed by centrifugation for 8 min at 225g at 4°C. Supernatant was subsequently removed and the resulting cell pellet resuspended in 500 μl of ice cold nuclei buffer (10mM Tris pH 7.6, 10mM NaCl, 2mM MgCl2, dH2O) containing protease inhibitors (Roche) for 10 min on ice. An equal volume of nuclei buffer/0.5% NP-40 was added to the tube and incubated for 5 min on ice. Samples were vortexed for 10 s and centrifuged for 1 min at 1,000g and 4°C. Cells were washed once in nuclei buffer/0.5% NP-40 containing protease inhibitors and centrifuged for 1 min at 1,000g and 4°C. Pellets were resuspended in 450µl dH2O and 60µl 10X restriction buffer (buffer 2 supplied with HindIII enzyme, New England BioLabs), incubated 1 hr with 15µl 10% SDS shaking at 900RPM at 37°C, and followed by an additional 1 hr incubation with 75µl 20% Triton X-100. 5 µl aliquots were taken as undigested controls and stored at 4°C. Samples were subsequently digested by adding 800U of HindIII (New England BioLabs) and incubating overnight at 37°C while shaking. 5 µl aliquots were taken as digested controls and de-crosslinked by incubation with 10 μl Proteinase K (10mg/ml, Roche) in 90 μl of 10 mM Tris (pH 7.5) at 65 °C for 1 h. Digestion efficiencies were estimated based on the pattern of smear of the undigested and digested controls by running 20 μl of decrosslinked sample on a 0.6% agarose gel. If digestion was sufficient, HindIII was inactivated by incubating the sample for 20 min at 65°C (shaking gently). The digested nuclei were transferred to a 50 ml falcon tube and mixed with 5.7ml dH2O, 700 μl 10X Ligase Buffer, and 50U T4 Ligase (Roche), and incubated overnight at 16°C. Ligation efficiency was determined by taking 100 μl of ligation reaction and incubating 1 hr at 65°C with 5 μl Proteinase K (10 mg/ml). When run in a 0.6% agarose gel, ligated DNA should appear as a single upper band similar to the undigested control. If ligation occurred, DNA crosslinks were reversed by adding 30 μl of 10 mg/ml Proteinase K and incubation at 65 °C overnight. Subsequently, 15 μl of 20 mg/ml PureLink RNase A (Invitrogen) was added and the reactions incubated for 45 min at 37 °C, followed by phenol extraction and DNA purification as described in Splinter et al, 2012. The DNA pellet was dissolved in 150 μl of 10 mM Tris (pH 7.5), and digested overnight with 50U DpnII (New England BioLabs) at 37°C while shaking. An aliquot of 5 μl was taken from the DpnII reaction and mixed with 95 μl of 10 mM Tris (pH 7.5), and 20 μl loaded into a 0.6% agarose gel to assess digestion efficiency. If sufficient digestion was achieved, DpnII was heat inactivated by incubating 20 minutes at 65°C, and DNA was ligated at low concentrations (12.1ml dH2O, 1.3ml 10X ligation buffer, 100U T4 DNA Ligase) overnight at 16°C. DNA was phenol extracted and ethanol precipitated with glycogen (Roche) as a carrier. The resulting 4C templates were purified using QIAquick PCR purification kit columns (Qiagen), dissolved in 10 mM Tris (pH 7.5), and stored at -20°C. Inverse 4C amplification primers were designed per viewpoint following standard rules for PCR primer design, and checking alignment uniqueness to the desired fragment as compared to the rest of the genome. Primers used in this study are listed in Supplementary Table 1c. Additionally, amplification primers for all viewpoints were added the PE1 and PE2 Illumina paired-end primers plus a 1-2 nucleotide barcode in their 5' ends for HiSeq PEx100 sequencing. Each of the 14 viewpoints was amplified from the available Df/+Bl6 and +129/+Bl6 4C templates in reactions using 3.2µg 4C template, 16µl dNTP (10mM, New England BioLabs), 24µl reading primer PE1 of a 1µg/µl primer stock, 24µl reading primer PE2 of a 1µg/µl primer stock, 11.2 µl Expand Long Template polymerase (Roche), 80µl 10X PCR buffer 1 (supplied with polymerase), and dH2O until completing 800µl total. This volume is then mixed and separated into 16x50µl PCR reactions, and run using the following program: 94 °C for 2 min, followed by 30 cycles of 15 s at 94 °C, 1 min at 55 °C and 3 min at 68 °C, and one final step of 5min at 68 °C. PCR reactions were subsequently collected and pooled together, and purified using the High Pure PCR Product Purification Kit (Roche) for viewpoints amplified on 129S5E71 and 129S5E117 4C templates, or using AMPure beads (Beckman Coulter) with a 0.9X volume ratio for viewpoints amplified from 129S5E98 and 129S5E118 4C templates. Equimolar amounts of isolated captured viewpoints were pooled together using the KK4824 kit to correct for insert size lengths (Kapa Biosystems). Pooled libraries were sequenced using two lanes of HiSeq 2000 PE100.