GSM3175722: 4C_Sik1_siEsrrb_Rep1; Mus musculus; OTHER
Crosslinked cells were lysed for 15 min on ice in 10 mM Tris-HCl pH 8.0, 10 mM NaCl, and 0.2% NP-40 supplemented with cOmplete protease inhibitor. Nuclei were isolated by centrifugation and by removing the supernatant. Nuclei corresponding to 10 million cells were resuspended in 500 μl of the primary restriction enzyme buffer. SDS was added to a final concentration of 0.3% and samples were incubated for 1 h at 37oC at 1,200 rpm on a thermo-mixer. SDS was quenched by addition of Triton X-100 to a final concentration of 3%. Primary restriction enzyme (400 units) was added and samples were digested for 6 h on a thermo-mixer followed by addition of 400 additional units of the primary restriction enzyme and overnight digestion. The primary restriction enzyme was inactivated by heating to 65oC for 20 min. Samples were diluted to a total volume of 8 ml in ligation buffer (66 mM Tris-HCl pH 7.5, 5 mM MgCl2, 5 mM DTT, 1 mM ATP). Proximity ligation was carried out by adding 4000 units of T4 DNA ligase and incubating at room temperature overnight. After reversal of -crosslinking and RNA removal, DNA was extracted by phenol/chloroform and purified by ethanol precipitation. A secondary digestion was performed overnight in a volume of 500 μl with 200 units of the secondary restriction enzyme. For secondary ligation, following inactivation of the restriction enzyme, each sample was diluted to 14 ml with ligation buffer. T4 DNA ligase (4000 units) was added and samples were incubated overnight at room temperature. DNA was extracted by phenol/chloroform and purified by ethanol precipitation. To remove salts, DNA was further purified using QIAquick PCR Cleanup kit. To construct 4C-seq libraries, reading and non-reading primers were designed for each viewpoint. PCR was performed in sixteen 25-μl PCR reactions using Platinum Taq DNA Polymerase High Fidelity. Reaction conditions were as in the manufacturer's instructions except 1.4 μM of each primer and 200-400 ng of template were used in each reaction. The PCR program was as follows: (1) 95oC, 5 min; (2) 95oC, 30 s; (3) 55oC, 1 min; (4) 68oC, 3 min; (5) go to (2), 30 cycles; (6) 68oC, 7 min. PCR products were first purified using illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) to remove primer dimers and then further purified using QIAquick PCR Purification Kit to remove salt. For ChIP-seq, crosslinked cells were resuspended in swelling buffer (25 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, and 1X cOmplete protease inhibitor cocktail [Roche]) and incubated for 10 min on ice. Cells were centrifuged and the cell pellet was resuspended in Buffer A (50 mM HEPES-KOH pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS) plus 1X cOmplete protease inhibitor cocktail. Cell sonication was performed on a Qsonica Q800R2 sonicator with 20% amplitude for 20 cycles at 10s each with 30s between cycles at 4oC. Sonicated lysate was pre-cleared by incubating with Dynabeads Protein A/G. Part of the pre-cleared lysate was used as input and the remainder was incubated overnight at 4°C with 2-10 micrograms of antibody. DNA/Protein-antibody conjugates were precipitated using Dynabeads Protein A/G blocked with 5 mg/ml BSA in PBS. Beads were washed twice each with Buffer A, Buffer B (50 mM HEPES-KOH pH 7.9, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), LiCl buffer (20 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% Na-deoxycholate, 0.5% NP-40) and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). DNA was eluted in elution buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS). Cross-links were reversed overnight at 65°C. RNA and protein were digested using RNase A and Proteinase K, respectively, and DNA was purified by phenol-chloroform extraction and ethanol precipitation. For mRNA-seq, total RNA was extracted from mESCs using TRIzol reagent (Invitrogen) according to manufacturer's instruction. To remove genomic DNA contamination, RNA samples were treated with DNaseI and purified again by phenol/chloroform extraction and ethanol precipitation. For Nascent RNA-seq, mESCs were harvested, washed with DPBS and lysed in 200 μl of ice-cold lysis buffer (10 mM Tris-HCl pH 7.5, 0.1% NP40, 150 mM NaCl). The cell lysate was gently layered over 500 μl of chilled sucrose cushion (24% RNAse-free sucrose in lysis buffer) in a new Eppendorf tube and centrifuged for 10 min at 4°C, 10,000xg. The supernatant (cytoplasmic fraction) was removed and the pellet (nuclei) was washed once with 200 μl of ice-cold 1 × PBS/1 mM EDTA. The nuclear pellet was resuspended in 100 μl of pre-chilled glycerol buffer (20 mM Tris-HCl pH 7.9, 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 0.125 mM PMSF, 50% glycerol) by gentle flicking of the tube. An equal volume (100 μl) of cold nuclear lysis buffer (10 mM HEPES pH 7.6, 1 mM DTT, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, 1% NP-40) was added. The mix was vortexed vigorously for 2 s. The sample was incubated for 2 min on ice, and then centrifuged for 2 min at 4°C, 10,000xg. The supernatant (nuclear fraction/nucleoplasm) was removed and the pellet (chromatin) was gently rinsed with ice-cold 1×PBS/1 mM EDTA. 1 ml of Trizol reagent was added to the chromatin and incubated for 30 min at 50oC to dissolve it. Nascent RNA was extracted following the manufacturer's instructions. For 4C-seq, crosslinked cells were lysed for 15 min on ice in 10 mM Tris-HCl pH 8.0, 10 mM NaCl, and 0.2% NP-40 supplemented with cOmplete protease inhibitor. Nuclei were isolated by centrifugation and by removing the supernatant. Nuclei corresponding to 10 million cells were resuspended in 500 μl of the primary restriction enzyme buffer. SDS was added to a final concentration of 0.3% and samples were incubated for 1 h at 37oC at 1,200 rpm on a thermo-mixer. SDS was quenched by addition of Triton X-100 to a final concentration of 3%. Primary restriction enzyme (400 units) was added and samples were digested for 6 h on a thermo-mixer followed by addition of 400 additional units of the primary restriction enzyme and overnight digestion. The primary restriction enzyme was inactivated by heating to 65oC for 20 min. Samples were diluted to a total volume of 8 ml in ligation buffer (66 mM Tris-HCl pH 7.5, 5 mM MgCl2, 5 mM DTT, 1 mM ATP). Proximity ligation was carried out by adding 4000 units of T4 DNA ligase and incubating at room temperature overnight. After reversal of -crosslinking and RNA removal, DNA was extracted by phenol/chloroform and purified by ethanol precipitation. A secondary digestion was performed overnight in a volume of 500 μl with 200 units of the secondary restriction enzyme. For secondary ligation, following inactivation of the restriction enzyme, each sample was diluted to 14 ml with ligation buffer. T4 DNA ligase (4000 units) was added and samples were incubated overnight at room temperature. DNA was extracted by phenol/chloroform and purified by ethanol precipitation. To remove salts, DNA was further purified using QIAquick PCR Cleanup kit. For ChIP-seq, libraries were prepared using a KAPA LTP kit following manufacturer's instructions. For mRNA-seq, libraries were prepared with KAPA stranded mRNA-Seq kits. For Nascent RNA-seq, libraries were constructed using KAPA Stranded RNA-Seq Kits with RiboErase (HMR). For 4C-seq libraries construction, reading and non-reading primers were designed for each viewpoint. PCR was performed in sixteen 25-μl PCR reactions using Platinum Taq DNA Polymerase High Fidelity. Reaction conditions were as in the manufacturer's instructions except 1.4 μM of each primer and 200-400 ng of template were used in each reaction. The PCR program was as follows: (1) 95oC, 5 min; (2) 95oC, 30 s; (3) 55oC, 1 min; (4) 68oC, 3 min; (5) go to (2), 30 cycles; (6) 68oC, 7 min. PCR products were first purified using illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) to remove primer dimers and then further purified using 
GEO Accession
GSM3175722