title: GSM2108996: E11.5_limb rep; Mus musculus; OTHER
library_construction_protocol: 10-20 million fixed cells were incubated for 15 min on ice in 200 ml of lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2% NP40, supplemented with fresh protease inhibitor cocktail).  Cells were then disrupted on ice with a dounce homogenizer (pestle B; 2 x 20 strokes), cell suspensions transferred to eppendorf tubes and centrifuged 5 min at 2000 g.  Supernatants were removed, the cell pellets washed twice with 100 ml of 1X CutSmart buffer (NEB) and the cell pellet resuspended in 100 ml of 1X CutSmart buffer, and divided into two eppendorf tubes.  1X CutSmart buffer (337 ml) was added to each tube, and the mixture was incubated 10 min at 65°C with 0.1% SDS.  44 ml of 10% Triton X-100 were added before overnight digestion with 400 Units HindIII.  The restriction enzyme was then inactivated by adding 86 ml of 10% SDS, and incubation for 30 min at 65°C.  Samples were then individually diluted into 7.62 ml of ligation mix (750 ml 10% Triton X-100, 750 ml 10X ligation buffer, 80 ml 10 mg/ml of BSA, 80 ml 100 mM ATP and 3000 Cohesive end Units of T4 DNA ligase) and incubated at 16°C for 2 hours. 3C libraries were incubated overnight at 65°C with 50 ml Proteinase K (10 mg/ml), and with an additional 50 ml Proteinase K the following day for 2 hours.  The DNA was purified by one phenol and one phenol-chloroform extraction, and precipitated with 0.1 volume (800 ml) of 3 M NaOAc pH 5.2 and 2.5 volumes of cold EtOH (20 ml).  After at least 1 h at -80°C, the DNA was centrifuged for 25 min at 20,000 g at 4°C, and the pellets washed with cold 70% EtOH. DNA was resuspended in 400 ml of TE pH 8.0, and transferred to eppendorf tubes for another phenol-chloroform extraction and precipitation with 40 ml of 3 M NaOAc pH 5.2 and 1.1 ml of cold EtOH.  DNA was recovered by centrifugation, and washed eight times with cold 70% EtOH.  Pellets were then dissolved in 100 ml TE pH 8.0, and incubated with RNAse A (1 ml at 10 mg/ml) for 15 min at 37°C. 3C libraries were denatured and annealed to 5C primers. 5C oligonucleotide were designed upstream (FORWARD) or downstream (REVERSE) EcoRI restriction sites. Annealing was followed by ligation with Taq DNA ligase. Ligated 5C primers were then amplified with the A-key and P1-key primers to generate the 5C libraries. 5C libraries were then prepared for sequencing onto Ion 316™ Chips with an Ion PGM™ Sequencer following either the Ion Xpress™ Template Kit v2.0, and Ion Sequencing Kit v2.0 protocols, or the Ion PGM™ Template OT2 200 Kit, and Ion PGM™ Sequencing 200 Kit v2.0 protocols as recommended by the manufacturer’s instructions (Life TechnologiesTM). Libraries were prepared according to Ion Torrent's instructions
attributes.tag: GEO Accession
attributes.value: GSM2108996