title: mmus_060313-5.6.7
organism: Mus musculus
description: 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. Livers from wildtype C57BL6/J male mice (approximately 12 months old) were obtained postmortem. 5 ug of antibody against the liver enriched transcription factor ONECUT1 (HNF6; Santa Cruz Biotechnology antibody sc13050) were used for each replicate. Individual 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 (doi:10.1016/j.ymeth.2009.03.001)). For each biological ChIP-exo replicate, livers from 3 male mice (approximately 12 months old) were pooled and approximately 1/4 of this pool was utilized. The ChIP-exo portion of the protocol was performed as previously described (doi: 10.1016/j.cell.2011.11.013) 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 was designed based on the adapter sequence provided by NEB: 5prime-Phos- TGACTGGAGTTCAGAGCTGTGCTCTTCCGATCT-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 65 C followed by RNaseA (Ambion) and ProteinaseK (Invitrogen) treatments. DNA was extracted using phenol-chloroform-isoamyl protocol and ethanol precipitation. DNA was denatured at 95 C, 3prime ends were primer-extended with a P7 fill-in primer (5prime-OH-AGACTGGAGTTCAGAGCTGTGCTCTTCCGATCT-OH-3prime), and phi29 polymerase. A second adaptor ligation to only _ exonuclease-digested ends of double stranded DNA was performed using 15 pmol of P5 adapter (sequence provided by NEB: 5primeOH-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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2014-03-15T17:00:37Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T16:53:31Z
attributes.tag: External Id
attributes.value: SAMEA2242750
attributes.tag: INSDC center name
attributes.value: SickKids Research Institute
attributes.tag: INSDC first public
attributes.value: 2014-03-15T17:00:37Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T16:53:31Z
attributes.tag: INSDC status
attributes.value: suppressed
attributes.tag: Submitter Id
attributes.value: E-MTAB-2060:mmus_060313-5.6.7
attributes.tag: age
attributes.value: 12 month
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type
attributes.tag: individual
attributes.value: 5,6,7
attributes.tag: organism part
attributes.value: liver
attributes.tag: sample name
attributes.value: E-MTAB-2060:mmus_060313-5.6.7
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL6/J