GSM7767573: Setd8-LKO2,TCPOBOP, input, ChIP-seq; Mus musculus; ChIP-Seq
For RNA: Total RNAs were purified from liver tissues by brief homogenization in 10 volumes of Trizol reagent using Polytron device, followed by incubation at room temperature for 5 min and the addition of 0.2 volumes of chloroform and further incubation at room temperature for 3 minutes. The samples were centrifuged at 12000 g for 15 minutes at 40C and the aqueous phase was collected, precipitated by the addition of equal volume of isopropanol. After 10 minutes at room temperature, the RNA was collected by centrifugation at 12000g for 15 minutes. The pellet was resuspended in H2O and re-precipitated with ethanol. The RNA samples were further purified by digestion with 10 units of DNase-I for 10 min at 370C, followed by purification with phenol/chlorophorm extraction and ethanol precipitation. For ChIP: Livers were minced to small pieces and crosslinked with 1% formaldehyde for 10 min. After the addition of glycine at 0.125 M final concentration, the cross-linked cells were washed with a buffer containing 50 mM Hepes pH 7.9, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and sequentially treated with a buffer containing 0.25% Triton-X100, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES and a buffer containing 0.15M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES for 10 minutes each. The resulting crosslinked nuclei were resuspended in 10 volumes of 50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 0.1% Na-deoxycholate, 0.5% Sarkosyl, and protease inhibitor cocktail (CompleteTM Roche), sonicated for 10 minutes in Covaris Sonicator instrument with maximum setting. After sonication, samples were supplemented with 0.5 volume of 50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 3% Triton X-100, 0.1% Sodium deoxycholate, and protease inhibitor cocktail, and centrifuged for 15 minutes at 20.000g. The soluble chromatin was incubated overnight with Dynabeads Protein G (Invitrogen), that were prebound by 5 mg of the primary antibodies recognizing H3K27ac-modified histones, CTCF and Smc3. The beads were washed twice with a buffer containing 50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 0.5 mM PMSF, 2 mg/ml aprotinin, once with the same buffer but containing 500 mM NaCl, once with a buffer containing 20 mM Tris, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5 % NP-40, 0.5 % Na-deoxycholate, 0.5 mM PMSF, 2 mg/ml aprotinin and once with a buffer containing 10 mM Tris pH 8.0, 0.1 mM EDTA, 0.5 mM PMSF, 2 mg/ml aprotinin.  Immunoprecipitated chromatin was eluted from the beads and reverse crosslinked by overnight incubation in a buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% SDS and 50 mM NaHCO3 at 65oC. The samples were then incubated with 20 mg/ml RNase-A for 30 minutes at 37oC, followed by incubation with 20 mg/ml Proteinase-K for 2 hours at 42oC. DNA was extracted by phenol/chlorophorm and precipitated with ethanol. For CUT&Tag and ATAC-seq: Hepatocyte nuclei were prepared as follows: liver tissue was minced into small pieces in 20 volumes of ice cold buffer containing 10 mM Tris pH 7.5, 2 mM MgCl2, 3 mM CaCl2 and protease inhibitor cocktail (CompleteTM EDTA-free, Roche), and homogenized by 10 strokes using Dounce device. The suspension was filtered through 100 m mesh strainer and centrifuged for 5 min. at 400g. The resulting pellet was resuspended in a buffer containing 10 mM Tris pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 10% glycerol, 1% NP40 and protease inhibitor cocktail (CompleteTM EDTA-free, Roche). After incubation in ice for 3 min the nuclei were collected by centrifugation for 5 min at 400g, washed twice with PBS and used for downstream applications. CUT&Tag assays were performed as follows: 105 nuclei were resuspended in a buffer containing 20 mM HEPES–KOH, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine, protease inhibitor cocktail (CompleteTM EDTA-free, Roche) and incubated for 10 min with Concanavalin A-conjugated magnetic beads (Epicypher) that were previously activated by repeated washings with a buffer containing 20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2. The reactions were supplemented with 0.5 volume of a buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail, 0.01% Digitonin, 2 mM EDTA and 0.5 g of the anti-Lamin A/C or anti-H4K20Me3 or IgG negative control primary antibodies (see Reporting Summary). The samples were incubated overnight at 40C by gentle agitation. The beads were collected and supplemented with a buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail, 0.01% Digitonin and 0.5 g of anti-rabbit or anti-mouse secondary antibodies (Epicypher). After incubation for 30 min at room temperature, the beads were washed twice with the same buffer and resuspended in the same buffer containing 300 mM NaCl. Protein A/G-fused Tn5 transposase protein (CUTANA pA/G-Tn5, Epicypher) was added and the samples were incubated for 1 hour at room temperature followed by washing with a buffer containing 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail, 0.01% Digitonin. The beads were collected and resuspended in the above buffer containing 10 mM MgCl2, and incubated for 1 hour at room temperature. The beads were collected and washed once with 10 mM TAPS, pH 8.5, 0.2 mM EDTA. DNA was released from the beads by incubation in 10 mM TAPS, pH 8.5, 0.1% SDS for 1 hour at 580C. After quenching by the addition of 3 volumes of 0.67% Triton-X100, the samples were used for PCR reaction (15 cycles) using universal i5 primers and barcoded i7 primers. For ATAC-seq reactions we used the ATAC-seq kit from Active Motif. Tagmentation reactions using 105 nuclei, DNA purification and PCR amplifications using combinations of indexed i5 and i7 primers were performed according to the manufacturer's instructions. RNA-seq libraries were generated using NEBNext Ultra II RNA Library preparation kit from New England Biolabs using Poly-A selection. ChIp-seq libraries were prepared using NEBNext Ultra II DNA Library Prep Kit of New England Biolabs. CUT&Tag and ATAC-seq libraries corresponded to the above-described PCR reactions with i5 and i7 primers, which were cleaned-up by AMPure.