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cell clones bearing the Atrx flox allele (1 × 107 cells) were transiently transfected with 50 μg of uncut Cre expression plasmid (pCAGGS-Cre-IRESpuro) [40]. Following transfection, cells were plated at a range of clonal densities in complete medium without G418, and isolated subclones were picked after 7 d. Subclones were expanded and analysed for the presence of a recombinant locus initially by PCR, to detect deletion of the MC1neopA cassette, and then by Southern blot and hybridisation with the intron 17 probe described above (Figure 2A and 2C). Northern blots were carried out according to standard techniques using 20 μg of total RNA isolated using TRI Reagent (Sigma-Aldrich, St. Louis, Missouri, United States). The blot was hybridised with a probe from within exon 10 of the Atrx gene (generated with primers Mxnp4 and Mxnp28 [Table S1]). After it was stripped, the membrane was hybridised with a β-actin cDNA probe (Clontech, Palo Alto, California, United States). Protein extraction and detection of Atrx by Western blotting was performed as described previously [4], using the mouse monoclonal anti-ATRX antibody 23C [15]. Analyses of cell cycle and apoptosis are described in Protocol S1. Methylation of rDNA was analysed in DNA from ES cell clones or from embryoid bodies recovered after 7 d of in vitro differentiation as described previously [41]. Genomic DNA was digested with methylation-sensitive restriction enzymes as described and analysed by Southern blotting. The RIB3 and RIB4 probes (which were amplified from human DNA, but cross-react with the mouse rDNA repeat) have been described previously [11]. Oligonucleotide probes to detect Line 1 and Sine B1 repeats have been described previously [42]. The minor satellite probe was a 27-mer oligonucleotide (mCENT2). The major satellite probe was a 27-mer oligonucleotide (DG27). The IAP probe was an ~400 bp PCR product (primers 14A and 13K) amplified from an IAP inserted into the mouse agouti gene [43] and was a gift from Peter Warnecke and Tim Bestor. The PCR product included the entire 5′ LTR of the IAP. All oligonucleotide sequences are shown in Table S1."}

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cell clones bearing the Atrx flox allele (1 × 107 cells) were transiently transfected with 50 μg of uncut Cre expression plasmid (pCAGGS-Cre-IRESpuro) [40]. Following transfection, cells were plated at a range of clonal densities in complete medium without G418, and isolated subclones were picked after 7 d. Subclones were expanded and analysed for the presence of a recombinant locus initially by PCR, to detect deletion of the MC1neopA cassette, and then by Southern blot and hybridisation with the intron 17 probe described above (Figure 2A and 2C). Northern blots were carried out according to standard techniques using 20 μg of total RNA isolated using TRI Reagent (Sigma-Aldrich, St. Louis, Missouri, United States). The blot was hybridised with a probe from within exon 10 of the Atrx gene (generated with primers Mxnp4 and Mxnp28 [Table S1]). After it was stripped, the membrane was hybridised with a β-actin cDNA probe (Clontech, Palo Alto, California, United States). Protein extraction and detection of Atrx by Western blotting was performed as described previously [4], using the mouse monoclonal anti-ATRX antibody 23C [15]. Analyses of cell cycle and apoptosis are described in Protocol S1. Methylation of rDNA was analysed in DNA from ES cell clones or from embryoid bodies recovered after 7 d of in vitro differentiation as described previously [41]. Genomic DNA was digested with methylation-sensitive restriction enzymes as described and analysed by Southern blotting. The RIB3 and RIB4 probes (which were amplified from human DNA, but cross-react with the mouse rDNA repeat) have been described previously [11]. Oligonucleotide probes to detect Line 1 and Sine B1 repeats have been described previously [42]. The minor satellite probe was a 27-mer oligonucleotide (mCENT2). The major satellite probe was a 27-mer oligonucleotide (DG27). The IAP probe was an ~400 bp PCR product (primers 14A and 13K) amplified from an IAP inserted into the mouse agouti gene [43] and was a gift from Peter Warnecke and Tim Bestor. The PCR product included the entire 5′ LTR of the IAP. All oligonucleotide sequences are shown in Table S1."}

{"project":"2_test","denotations":[{"id":"16628246-12085230-85799785","span":{"begin":155,"end":157},"obj":"12085230"},{"id":"16628246-14729260-85799786","span":{"begin":1082,"end":1083},"obj":"14729260"},{"id":"16628246-10570185-85799787","span":{"begin":1137,"end":1139},"obj":"10570185"},{"id":"16628246-8417345-85799788","span":{"begin":1366,"end":1368},"obj":"8417345"},{"id":"16628246-10742099-85799789","span":{"begin":1630,"end":1632},"obj":"10742099"},{"id":"16628246-11711429-85799790","span":{"begin":1727,"end":1729},"obj":"11711429"},{"id":"16628246-7926745-85799791","span":{"begin":1982,"end":1984},"obj":"7926745"},{"id":"T83163","span":{"begin":155,"end":157},"obj":"12085230"},{"id":"T14123","span":{"begin":1082,"end":1083},"obj":"14729260"},{"id":"T85420","span":{"begin":1137,"end":1139},"obj":"10570185"},{"id":"T26504","span":{"begin":1366,"end":1368},"obj":"8417345"},{"id":"T27595","span":{"begin":1630,"end":1632},"obj":"10742099"},{"id":"T53645","span":{"begin":1727,"end":1729},"obj":"11711429"},{"id":"T82024","span":{"begin":1982,"end":1984},"obj":"7926745"}],"text":"ES cell clones bearing the Atrx flox allele (1 × 107 cells) were transiently transfected with 50 μg of uncut Cre expression plasmid (pCAGGS-Cre-IRESpuro) [40]. Following transfection, cells were plated at a range of clonal densities in complete medium without G418, and isolated subclones were picked after 7 d. Subclones were expanded and analysed for the presence of a recombinant locus initially by PCR, to detect deletion of the MC1neopA cassette, and then by Southern blot and hybridisation with the intron 17 probe described above (Figure 2A and 2C). Northern blots were carried out according to standard techniques using 20 μg of total RNA isolated using TRI Reagent (Sigma-Aldrich, St. Louis, Missouri, United States). The blot was hybridised with a probe from within exon 10 of the Atrx gene (generated with primers Mxnp4 and Mxnp28 [Table S1]). After it was stripped, the membrane was hybridised with a β-actin cDNA probe (Clontech, Palo Alto, California, United States). Protein extraction and detection of Atrx by Western blotting was performed as described previously [4], using the mouse monoclonal anti-ATRX antibody 23C [15]. Analyses of cell cycle and apoptosis are described in Protocol S1. Methylation of rDNA was analysed in DNA from ES cell clones or from embryoid bodies recovered after 7 d of in vitro differentiation as described previously [41]. Genomic DNA was digested with methylation-sensitive restriction enzymes as described and analysed by Southern blotting. The RIB3 and RIB4 probes (which were amplified from human DNA, but cross-react with the mouse rDNA repeat) have been described previously [11]. Oligonucleotide probes to detect Line 1 and Sine B1 repeats have been described previously [42]. The minor satellite probe was a 27-mer oligonucleotide (mCENT2). The major satellite probe was a 27-mer oligonucleotide (DG27). The IAP probe was an ~400 bp PCR product (primers 14A and 13K) amplified from an IAP inserted into the mouse agouti gene [43] and was a gift from Peter Warnecke and Tim Bestor. The PCR product included the entire 5′ LTR of the IAP. All oligonucleotide sequences are shown in Table S1."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T11443","span":{"begin":0,"end":7},"obj":"CL:0002322"},{"id":"T11444","span":{"begin":27,"end":31},"obj":"PR:000004503"},{"id":"T11445","span":{"begin":32,"end":36},"obj":"SO:0000359"},{"id":"T11446","span":{"begin":37,"end":43},"obj":"SO:0001023"},{"id":"T11447","span":{"begin":77,"end":88},"obj":"GO:0009294"},{"id":"T11448","span":{"begin":105,"end":108},"obj":"MOP:0000780"},{"id":"T11449","span":{"begin":113,"end":123},"obj":"GO:0010467"},{"id":"T11450","span":{"begin":124,"end":131},"obj":"SO:0000155"},{"id":"T11451","span":{"begin":144,"end":148},"obj":"SO:0000243"},{"id":"T11452","span":{"begin":170,"end":182},"obj":"GO:0009294"},{"id":"T11453","span":{"begin":260,"end":264},"obj":"CHEBI:42768"},{"id":"T11454","span":{"begin":442,"end":450},"obj":"SO:0005853"},{"id":"T11455","span":{"begin":482,"end":495},"obj":"GO:0097617"},{"id":"T11456","span":{"begin":505,"end":511},"obj":"SO:0000188"},{"id":"T11457","span":{"begin":666,"end":673},"obj":"CHEBI:33893"},{"id":"T11458","span":{"begin":740,"end":750},"obj":"GO:0097617"},{"id":"T11459","span":{"begin":776,"end":780},"obj":"SO:0000147"},{"id":"T11460","span":{"begin":791,"end":795},"obj":"PR:000004503"},{"id":"T11461","span":{"begin":796,"end":800},"obj":"SO:0000704"},{"id":"T11462","span":{"begin":817,"end":824},"obj":"SO:0000112"},{"id":"T11463","span":{"begin":895,"end":905},"obj":"GO:0097617"},{"id":"T11464","span":{"begin":913,"end":920},"obj":"PR:000003676"},{"id":"T11465","span":{"begin":1018,"end":1022},"obj":"PR:000004503"},{"id":"T11466","span":{"begin":1096,"end":1101},"obj":"NCBITaxon:10088"},{"id":"T11467","span":{"begin":1118,"end":1122},"obj":"PR:000004503"},{"id":"T11468","span":{"begin":1123,"end":1131},"obj":"GO:0042571"},{"id":"T11469","span":{"begin":1154,"end":1164},"obj":"GO:0007049"},{"id":"T11470","span":{"begin":1169,"end":1178},"obj":"GO:0006915"},{"id":"T11471","span":{"begin":1254,"end":1261},"obj":"CL:0002322"},{"id":"T11472","span":{"begin":1277,"end":1292},"obj":"UBERON:0014374"},{"id":"T11473","span":{"begin":1371,"end":1378},"obj":"SO:0001026"},{"id":"T11474","span":{"begin":1543,"end":1548},"obj":"NCBITaxon:9606"},{"id":"T11475","span":{"begin":1579,"end":1584},"obj":"NCBITaxon:10088"},{"id":"T11476","span":{"begin":1886,"end":1888},"obj":"SO:0000028"},{"id":"T11477","span":{"begin":1889,"end":1900},"obj":"SO:0000006"},{"id":"T11478","span":{"begin":1902,"end":1909},"obj":"SO:0000112"},{"id":"T11479","span":{"begin":1963,"end":1968},"obj":"NCBITaxon:10088"},{"id":"T11480","span":{"begin":1969,"end":1975},"obj":"PR:000004372"},{"id":"T11481","span":{"begin":1976,"end":1980},"obj":"SO:0000704"},{"id":"T11482","span":{"begin":2041,"end":2052},"obj":"SO:0000006"}],"text":"ES cell clones bearing the Atrx flox allele (1 × 107 cells) were transiently transfected with 50 μg of uncut Cre expression plasmid (pCAGGS-Cre-IRESpuro) [40]. Following transfection, cells were plated at a range of clonal densities in complete medium without G418, and isolated subclones were picked after 7 d. Subclones were expanded and analysed for the presence of a recombinant locus initially by PCR, to detect deletion of the MC1neopA cassette, and then by Southern blot and hybridisation with the intron 17 probe described above (Figure 2A and 2C). Northern blots were carried out according to standard techniques using 20 μg of total RNA isolated using TRI Reagent (Sigma-Aldrich, St. Louis, Missouri, United States). The blot was hybridised with a probe from within exon 10 of the Atrx gene (generated with primers Mxnp4 and Mxnp28 [Table S1]). After it was stripped, the membrane was hybridised with a β-actin cDNA probe (Clontech, Palo Alto, California, United States). Protein extraction and detection of Atrx by Western blotting was performed as described previously [4], using the mouse monoclonal anti-ATRX antibody 23C [15]. Analyses of cell cycle and apoptosis are described in Protocol S1. Methylation of rDNA was analysed in DNA from ES cell clones or from embryoid bodies recovered after 7 d of in vitro differentiation as described previously [41]. Genomic DNA was digested with methylation-sensitive restriction enzymes as described and analysed by Southern blotting. The RIB3 and RIB4 probes (which were amplified from human DNA, but cross-react with the mouse rDNA repeat) have been described previously [11]. Oligonucleotide probes to detect Line 1 and Sine B1 repeats have been described previously [42]. The minor satellite probe was a 27-mer oligonucleotide (mCENT2). The major satellite probe was a 27-mer oligonucleotide (DG27). The IAP probe was an ~400 bp PCR product (primers 14A and 13K) amplified from an IAP inserted into the mouse agouti gene [43] and was a gift from Peter Warnecke and Tim Bestor. The PCR product included the entire 5′ LTR of the IAP. All oligonucleotide sequences are shown in Table S1."}