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n2:pmcid
PMC0
bibo:doi
10.1038%2Fnature11043
n6:contains
_:vb16663534 _:vb16663554
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_:vb16663534
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n6:Section
dc:title
methods
n6:contains
_:vb16663552 _:vb16663553 _:vb16663548 _:vb16663549 _:vb16663550 _:vb16663551 _:vb16663544 _:vb16663545 _:vb16663546 _:vb16663547 _:vb16663540 _:vb16663541 _:vb16663542 _:vb16663543 _:vb16663536 _:vb16663537 _:vb16663538 _:vb16663539 _:vb16663535
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_:vb16663535
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ELK4 knockdown target sequences are as follows: ELK4 KD1, 5’-CGACACAGACATTGATTCA-3’; ELK4 KD2, 5’-GAGAATGGAGGGAAAGATA-3’, as previously described>>20<<. ELK1 knockdown target sequence: GATGTGAGTAGAAGAGTTA. GABPα knockdown target sequence: TGAAGAAGCTCAAGTGATA. HDAC1 knockdown target sequences: HDAC1 KD1 5’- AGAAAGACCCAGAGGAGAA-3’, HDAC1 KD2 5’-GCAAGCAGATGCAGAGATT-3’. Double-stranded
n2:mentions
n3:18469865
Subject Item
_:vb16663536
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n2:Context
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Anchorage-independent growth was measured as previously described>>30<<. Annexin V analysis was performed using the FITC Annexin Apoptosis Detection Kit (BD Pharmingen), on S7KD and control U2OS cells cultured in 1% serum.
n2:mentions
n3:16054100
Subject Item
_:vb16663537
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Samples enriched for cytoplasmic, nucleoplasmic, and chromatin fractions were prepared as previously described>>31<<. Co-immunoprecipitations were performed as previously described32, except that one 150 mm plate of cells was used per IP, Protein A/G beads (Sigma) were used instead of FLAG-resin, and elution was performed by boiling beads in Laemmli
n2:mentions
n3:11046155
Subject Item
_:vb16663538
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n2:Context
rdf:value
Co-immunoprecipitations were performed as previously described>>32<<, except that one 150 mm plate of cells was used per IP, Protein A/G beads (Sigma) were used instead of FLAG-resin, and elution was performed by boiling beads in Laemmli loading buffer.
n2:mentions
n3:19187765
Subject Item
_:vb16663539
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Purification of human SIRT7 protein from baculovirus-infected insect cells was described previously>>8<<. Calf thymus histones were obtained from Roche, and poly-nucleosomes were purified from HeLa cells as previously described33. Histone peptides were synthesized at the Yale W. M. Keck peptide synthesis facility, and liquid chromatography
n2:mentions
n3:16079181
Subject Item
_:vb16663540
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n2:Context
rdf:value
Calf thymus histones were obtained from Roche, and poly-nucleosomes were purified from HeLa cells as previously described>>33<<. Histone peptides were synthesized at the Yale W. M. Keck peptide synthesis facility, and liquid chromatography mass spectrometry was performed at the Stanford University Vincent Coates Foundation Mass Spectrometry Laboratory. To
n2:mentions
n3:16728974
Subject Item
_:vb16663541
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n2:Context
rdf:value
Acid-extracted total histones were subjected to chemical derivatization using D0-proionic anhydride and digestion with trypsin at a substrate to enzyme ratio of 10:1 for 6 hours at 37°C as previously described>>34<<. An additional round of propionylation was performed on the digested peptides, with one sample being derivatized with the same D0-propionic anhydride reagent, and the other being derivatized with D10-propionic anhydride for quantitative
n2:mentions
n3:17446892
Subject Item
_:vb16663542
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was performed on the digested peptides, with one sample being derivatized with the same D0-propionic anhydride reagent, and the other being derivatized with D10-propionic anhydride for quantitative proteomics as previously described>>35<<. D10-propionic anhydride introduces a 5 Da shift by derivatization of the free N-termini of all peptides generated from the trypsin digest.
n2:mentions
n3:19764812
Subject Item
_:vb16663543
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n2:Context
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Equal amounts of both samples as quantified earlier by a Bradford assay were mixed together, and digested peptides were desalted using homemade STAGE tips as reported earlier>>36<<. Desalted peptides were loaded onto fused silica microcapillary column (75 µm) packed with C18 resin constructed with an ESI tip through an Eksigent AS-2 autosampler (Eksigent Technologies Inc., Dublin, CA) at a rate of ~200 nL/minute.
n2:mentions
n3:12585499
Subject Item
_:vb16663544
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n2:Context
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ChIP for ChIP-sequencing analysis was performed as previously described>>37<<. Four ChIP samples were sequenced using Illumina Solexa Genome Analyzer II single end sequencing protocol, including two SIRT7 replicates and two input control replicates.
n2:mentions
n3:20418883
Subject Item
_:vb16663545
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The uniquely mapped reads from replicates of SIRT7 and input control samples were pooled respectively and processed by MACS (version 1.3.6)>>38<< to generate the whole-genome ChIP-sequencing profiles, with the “--diag” option enabled for the sequencing depth saturation test.
n2:mentions
n3:18798982
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_:vb16663546
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The SIRT7 target genes were identified by detecting the SIRT7 binding peaks within 3 kb upstream to 3 kb downstream of transcription start sites (TSS’s) of RefSeq genes using CEAS>>35<<. In total, 253 target genes were identified, including 241 protein-coding genes. The GO analysis was performed using DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov) 39,40.
n2:mentions
n3:19764812
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_:vb16663547
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The GO analysis was performed using DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov) >>39<<,40.
n2:mentions
n3:12734009
Subject Item
_:vb16663548
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The GO analysis was performed using DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov) 39,>>40<<.
n2:mentions
n3:19131956
Subject Item
_:vb16663549
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For identification of ELK4 ChIP-sequencing target genes, the ChIP-sequencing reads from O'Geen et al. (GSE24685)>>16<<, were remapped to hg18, and peaks were called using MACS (Supplementary Table 9).
n2:mentions
n3:21126370
Subject Item
_:vb16663550
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n2:Context
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De novo motifs with sizes from 6 to 15 nucleotides were searched within SIRT7 binding sites using MDModule>>41<<, with repetitive regions masked and running parameters “-s 100 -t 50”. The top 50 detected de novo motifs (top 5 of each motif size) were recorded and compared with JASPAR motif database using STAMP with default settings42.
n2:mentions
n3:12101404
Subject Item
_:vb16663551
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n2:Context
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The top 50 detected de novo motifs (top 5 of each motif size) were recorded and compared with JASPAR motif database using STAMP with default settings>>42<<. The position weight matrix of the ELK4 motif (Supplementary Table 6) was remapped to the identified SIRT7 peaks using cisgenome34, with parameter “–r 30”.
n2:mentions
n3:17478497
Subject Item
_:vb16663552
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n2:Context
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The position weight matrix of the ELK4 motif (Supplementary Table 6) was remapped to the identified SIRT7 peaks using cisgenome>>34<<, with parameter “–r 30”.
n2:mentions
n3:17446892
Subject Item
_:vb16663553
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Cells were prepared for ChIP as previously described>>29<<, with the exception that DNA was washed and eluted using a PCR purification kit (Qiagen) rather than by phenol-chloroform extraction.
n2:mentions
n3:17272500
Subject Item
_:vb16663554
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n6:Section
dc:title
methods summary
n6:contains
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Recombinant human SIRT7 protein was purified from baculovirus-infected insect cells as described>>8<<.
n2:mentions
n3:16079181
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_:vb16663556
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ChIP was performed as previously described>>29<<, except that the Qiagen PCR purification kit was used for DNA purification (Qiagen).
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