characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-05-06T17:00:43Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T17:31:10Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2501116
characteristics.characteristic: External Id
characteristics.text: SickKids Research Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2014-05-06T17:00:43Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T17:31:10Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS450997
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2060:mmus_3
characteristics.characteristic: Submitter Id
characteristics.text: 4 month
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UO_0000035
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: doi:10.1016/j.ymeth.2009.03.001; Shortly, liver material was crosslinked in 1% formaldehyde for 20 minutes followed by a 10 minute incubation in 500 mM glycine buffer to neutralize the formaldehyde. Samples were rinsed with PBS and frozen until use. Formaldehyde-crosslinked/flash frozen tissue was stored at -80C. doi:10.1016/j.ymeth.2009.03.001; Crosslinked animal livers were dounce homogenized into a single cell suspension and rinsed with ice cold PBS. Each pellet of cross-linked material was resuspended in 10 ml of LB1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; 1mM EDTA; 10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100) and pelleted again by centrifugation at 2,000 x rcf for 4 minutes at 4 degrees C. Pellets were then rinsed in 10 ml of LB2 (10 mM Tris-HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA) and pelleted again by centrifugation at 2,000 x rcf for 4 minutes at 4 degrees C. The nuclei preparation was resuspended in 3 ml LB3 (10 mM Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine). Chromatin in the nuclei fraction was fragmented to an average length of 300 bp by sonication. After sonication 300 ul of 10% Triton X-100 was added to the 3 ml of sonicated lysate and debris was removed by centrifugation at 20,000 x rcf for 10 minutes at 4 degrees C. For each of three biological ChIP-exo replicates, post mortem livers from 3 male C57BL/6J mice, approximately 4 months old, were used. These mice were maintained in specific pathogen-free conditions at the Hospital for Sick Children Laboratory Animal Services according to an approved animal use protocol and were kindly provided by Dr. Jayne Danska. Approximately 1/3 of each liver was utilized per ChIP-exo experiment. 7 ug of an antibody against the liver enriched transcription factor ONECUT1 (HNF6; Santa Cruz Biotechnology antibody sc13050) was used for each biological replicate. Livers were cross-linked in 1% formaldehyde and chromatin immunoprecipitation (ChIP) was performed as previously described (up until and including the RIPA buffer washes; step 38 from Schmidt et al. 2009) (32). The ChIP-exo portion of the protocol was performed as previously described (Rhee and Pugh 2011) with modifications making the assay compatible with the Illumina sequencing platform. Briefly, while still on the magnetic beads (Invitrogen, Dynabeads), the immunoprecipitated DNA was end-repaired (NebNext End Repair Module, New England Biolabs (NEB)). The P7 adapter (150 pmol) was designed based on the adapter sequence provided by NEB: 5prime-Phos-TGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-OH-3prime and 5prime-OH-AGATCGGAAGAGCACACGTCTGAACTCC-OH-3prime. After ligation, the DNA was nick-repaired with phi29 polymerase, and digested by lambda and RecJf exonucleases (NEB). DNA samples were eluted from the beads by performing reverse cross-linking overnight at 65C, followed by RNaseA (Ambion) and ProteinaseK (Invitrogen) treatments. DNA was extracted using a phenol-chloroform-isoamyl protocol and ethanol precipitation. DNA was denatured at 95C, and 3 prime ends were primer-extended with a P7 fill-in primer (5 pmol; 5prime-OH-TGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-OH-3prime) and phi29 polymerase. A second adaptor ligation to only lamda exonuclease-digested ends of double stranded DNA was performed using 15 pmol of P5 adapter (sequence provided by NEB: 5prime-OH-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTG-OH-3prime and 5prime-OH-TCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-OH-3prime). The resulting DNA samples were PCR-amplified using multiplexing index primers (NEBNext DNA Library Prep Master Mix Set for Illumina).
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: wild type genotype
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
characteristics.characteristic: genotype
characteristics.text: 3
characteristics.characteristic: individual
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: liver
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000384
characteristics.characteristic: sex
characteristics.text: C57BL/6J
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000606
characteristics.characteristic: strain
characteristics.text: mmus_3
characteristics.characteristic: title