characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-12-30T00:24:38Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2022-12-30T00:24:38Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA111497746
characteristics.characteristic: External Id
characteristics.text: Centenary Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2022-12-30T00:24:38Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-12-30T00:24:38Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS13591511
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12320:SOX7-V5 E9.5
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Sox7 v5 embryos at E9.5 and E10.5 were harvested in cold PBS, minced and subsequently digested in 0.05% trypsin/EDTA at 37°C for 10 mins. To isolate single cells, samples were passed in a 21 and 23 gauge needles. To cross link DNA-protein, samples were next treated with 1% formaldehyde buffer containing 50 mmol/L Hepes-KOH, 0.1 mol/L NaCl, 1 mM EDTA, 0.5 mmol/L EGTA for 10 mins with constant agitation at room temperature. Samples were then quenched with 1M glycine at pH7.5, fr 5 mins at room temperature, before washed again in PBS/10% FCS and rinsed subsequently in cold PBS. Cell pellet from each litter were snap freeze on dry ice, and stored in -80°C freezer. Cell pellets were thawed, and pooled together before cell lysis. Samples were sonicated, and 50ul from each sonication sample were set aside as the input DNA control. Samples were then incubated with 10ug V5 monoclonal antibody (R960CUS, Invitrogen). Following immunoprecipitation, the DNA for both input DNA control and samples were amplified using the TruSeq ChIPseq kit (Illumina, IP-202-1012) with 0.5 μmol/L of the universal forward and reverse PCR primer containing the index sequence of choice in 50 μL 1 x NEB Next High-Fidelity PCR Master Mix (New England Biolabs, M0541). Depending on the PCR efficiency, PCR cycles were conducted between 13 to 18 cycles. PCR product was finally purified with AMPure beads (1.8 volume) and eluted in 20 μL of resuspension buffer (Tris-Acetate 10 mmol/L pH8).
characteristics.characteristic: description
characteristics.text: embryonic day 9.5
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: whole organism
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: C57BL/6
characteristics.characteristic: strain
characteristics.text: SOX7-V5 E9.5
characteristics.characteristic: title