title: GSM3682120: ChipSeq_RawData_T6_Rep1; Mus musculus; ChIP-Seq
library_construction_protocol: RNA-Seq: Total RNA from 3 replicates (i.e. livers from 3 mice at each CT and from NS mice; see above) was extracted from liver pieces using the RNeasy Mini Kit (Qiagen). DNase treatment was performed with RQ1 RNase-Free DNase (M101, Promega). ChiP-Seq: ChIP of LMNB1 was done as described {Rønningen et al 2015 Genome Res 25: 1825-1835}. Snap-frozen liver tissue pieces (40-50 mg) were thawed on ice and minced on ice for 30 sec. Minced tissue was resuspended in PBS with 1 mM PMSF and protease inhibitors, and homogenized by 7-10 strokes in a 2-ml Dounce homogenizer using pestle 'B'. Samples were centrifuged at 400 g and supernatants discarded. Pellets were resuspended in PBS containing 1% formaldehyde and cross-linking allowed to occur for 10 min at room temperature. Cross-linked samples were sedimented and lysed in RIPA buffer (140 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 1% SDS, 0.1% sodium deoxycholate, 1 mM PMSF, protease inhibitors). To generate 200-500 bp DNA fragments, cells were sonicated 4 times 10 min in a Bioruptor (Diagenode). After sedimentation, the supernatant was diluted 10-fold in RIPA and incubated with anti-LMNB1 antibodies (10 µg; Abcam ab16048) coupled to Dynabeads Protein G (Invitrogen) overnight at 4oC. ChIP samples were washed 3 times in ice-cold RIPA. Cross-links were reversed and DNA eluted for 6 h at 68°C in elution buffer (50 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% SDS) containing 40 ng/ml proteinase K. DNA was extracted and libraries prepared and sequenced on an Illumina HiSeq2500. ChIP-seq and RNA-seq sequencing libraries were prepared according to  Illumina protocols for sequencing on a HSeq2500 at the Norwegian Sequencing Center (Oslo University Hospital).
attributes.tag: GEO Accession
attributes.value: GSM3682120