organism: Mus musculus
description: 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-30
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-30
attributes.tag: External Id
attributes.value: SAMEA111497749
attributes.tag: INSDC center alias
attributes.value: Centenary Institute
attributes.tag: INSDC center name
attributes.value: Centenary Institute
attributes.tag: INSDC first public
attributes.value: 2022-12-30T00:24:38Z
attributes.tag: INSDC last update
attributes.value: 2022-12-30T00:24:38Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12320:SOX7-V5 E9.5 input
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryonic day 9.5
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-12320:SOX7-V5 E9.5 input
attributes.tag: strain
attributes.value: C57BL/6