GSM1847805: Mutant.High.Whole.Rep1.F2; Mus musculus; MNase-Seq
Two weeks after AAV-Cre injection, which was previously shown to yield efficient deletion (Lee et al., 2014), livers were perfused with 45 ml of 37°C Liver Perfusion Medium (Life technologies, 17701-038), followed by 45 ml of 37°C Liver Digestion Medium (Life technologies, 17703-034) at a speed of 5 ml /min.  The perfused liver was excised, washed with SST (150 mM NaCl, 15 mM Na citrate, 10 mM Tris [pH 7.4]), and minced using cell scrapers to release the hepatocytes.  The hepatocytes were filtered through a 100 mm cell strainer into a 50 ml conical tube, which was then filled up to 50 ml with SST.  Hepatocytes were centrifuged at 50 g for 3 min at 4°C to pellet only hepatocyte cells.  The supernatant was aspirated and the pellet was resuspended in 50 ml SST.  Hepatocytes were centrifuged at 50 g for 3 min at 4°C.  The SST wash was repeated.  Hepatocytes were resuspended in 5 ml RSB (10 mM Tris [pH7.4], 10 mM NaCl, 3 mM MgCl2, 10 mM sodium butyrate, 0.5 mg/ml aprotinin, 0.5 mg/ml leupeptin, 1 mg/ml pepstatin) plus 0.5% NP40 and were swollen on ice for 5 min.  Liver and spleen from C3H mice were minced with scalpels in SST (150 mM NaCl, 15 mM Na Citrate, 10 mM Tris [pH 7.4]) on ice and washed with SST in 30 ml Corex tubes three times.  The tissue was resuspended in RSB (10 mM Tris [pH7.4], 10 mM NaCl, 3 mM MgCl2 with 10 mM sodium butyrate, 0.5 mg/ml aprotinin, 0.5 mg/ml leupeptin, 1 mg/ml pepstatin) and 0.5% NP40, and was swollen on ice for 5 min (liver) or 10 min (spleen).  The tissue was homogenized with a glass Teflon pestle in a glass homogenizer and a portion was observed under the microscope to confirm nuclear isolation. The nuclear suspension was filtered through a 100 mm cell strainer and pelleted by centrifugation at 1.4 K rpm for 7 min at 4°C in 15 ml Corex tubes with a swinging bucket rotor.  The pellet was washed in RSB plus 0.5% NP40 and pelleted by centrifugation at 900 rpm for 10 min at 4°C in 15 ml Corex tubes with a swinging bucket rotor; this wash step was repeated once.  The nuclear pellet was re-suspended in RSB and adjusted to O.D. A260 of 1 for liver and 0.68 for spleen.  CaCl2 (3 mM final) was added, the nuclear suspension was pre-warmed for 1.5 min at 37°C, and was treated with MNase (Worthington Biochemicals) at 0 U/ml for control, 0.5 or 1 U/ml for low digestion and 20 U/ml for high digestion for 2 min at 37°C.  Quench control (“Q” sample) to assess endogenous nucleases was without MNase and CaCl2 and was not warmed.  The reactions were stopped by adding an equal volume of 2x TNESK (20 mM Tris [pH7.4], 200 mM NaCl, 2 mM EDTA, 2% SDS, 0.2 mg/ml Proteinase K), and incubated overnight at 37 °C.  DNA was purified using phenol-chloroform extraction followed by ethanol precipitation.  The nucleic acid was digested with 10 ng/ml of RNase for 20 min at 37°C and DNA was purified using phenol-chloroform extraction followed by ethanol precipitation.  50 ug samples of low MNase and 10 ug samples of high MNase were run on 6% polyacrylamide gels (TBE).  The mono-nucleosomal DNA was excised from the 140 bp~200 bp size range and sub-nucleosomal DNA was excised from 25-140 bp size range.  The gel was sliced into small pieces, put in 2 vol of diffusion buffer (500 mM ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% SDS), and rotated overnight. The supernatant was filtered through Whatman GF/C and ethanol precipitated.  DNA was purified using QIA EX II (QIAGEN) for sub-nucleosomal DNA and AMPure XP (Beckman) beads for mono-nucleosomal DNA. Mono-nucleosomal DNA from low MNase digestion averaged 1.5~2.5% of bulk DNA. Mono-nucleosomal DNA from high MNase digestion averaged 25~30% of bulk DNA.  The percent of mononucleosomal DNA of bulk DNA was estimated based on the amount of mononucleosomal DNA recovered from a preparative PAGE gel, multiplied by 1.25 (PAGE gel extraction recovers about 80% of sample in our hands) compared to the amount of bulk DNA loaded onto to the PAGE gel. We prepared multiplexed libraries from two biological replicates of low and high MNase digested mono-nucleosomal DNA from WT liver, low and high MNase digested mono/sub-nucleosomal DNA from FoxA1/FoxA2 flox;AAV-Cre and WT;AAV-Cre.  The standard Illumina multiplexing library preparation protocol was employed with the following changes: 1) All QIAGEN purification steps were substituted with AMPure XP bead (Beckman) purifications. 2) For MNase digested mono-nucleosomal DNA, gel purification following adapter ligation was substituted with AMPure XP bead purification in order to remove excess adapters. 3) 10 ng of adapter ligated DNA was used as a template for PCR, wth 10 PCR cycles for MNase DNA, and 12 PCR cycles for ChIP DNA. 4) Gel purification after PCR was substituted with AMPure XP beads purification in order to remove excess primers.  The DNA libraries were assessed using an Agilent Technologies 2100 Bioanalyzer.  MNase-seq libraries from FoxA1/FoxA2 flox;AAV-Cre and WT;AAV-Cre were were sequenced as 37-bp paired-end by Illumina NextSeq500.
GEO
GSM1847805