description: Protocols: Breast tumors were dissected immediately after euthanizing the mice and stored at -80˚C. 50-100µg of frozen tissue was homogenized for in a powerlyzer24 (Mo Bio) in 500-1ml Qiazol(Qiagen) 3 times for 10 seconds at 4000 RPM, allowing samples to cool on ice between homogenization steps. Samples were then centrifuged on full speed for 30 seconds and the supernatant incubated for 10 minutes at room temperature to allow for dissociation of nucleotide complexes. 200µl Chloroform was added to the lysates and vortexed for 15 seconds.Samples were again incubated for 10 minutes, centrifuged for 10 minutes at 12000g, 4˚C and the upper phase transferred to new tubes. The chloroform step was then repeated to improve RNA quality. To the resulting upper phase, 250-500µl isopropanol was added and incubated for 30 minutes on ice. Samples were again centrifuged for 10 minutes at 12000g, 4˚C. Pellets were washed with 1ml of cold 70% EtOH and allowed to dry. Samples were then resuspended in 50µl Rnase free H2O. RNA-concentration was determined with a photometer (Denovix DS11+). RNA quality was checked via Agarose gel electrophoresis (1% agarose gel in TBE buffer, 5µl HD-green100ml gel (Intas). 400ng of RNA was mixed with 6X RNA loading dye to a final volume of 10µl. The agarose gel was run at 100V for 15 minutes. The efficiency of the Usp22fl/fl was validated by qPCR (see protocol below for cDNA synthesis and qPCR). RNA quality was further validated using RNA 6000 nano (Bioanalyzer, Aglient). RNA samples were prepared according to the TruSeq RNA sample preparation guide (Illumina) to a final library pool concentration of 2nM. Library concentration was quantified using a Qubit fluorometer (Invitrogen).
attributes.tag: ENA first public
attributes.value: 2021-05-26
attributes.tag: ENA last update
attributes.value: 2021-05-26
attributes.tag: External Id
attributes.value: SAMEA7050477
attributes.tag: INSDC center alias
attributes.value: University Medical Center Gottingen Clinic for General, Visceral and Pediatric Surgery Tumor Epigenetics
attributes.tag: INSDC center name
attributes.value: University Medical Center Gottingen Clinic for General, Visceral and Pediatric Surgery Tumor Epigenetics
attributes.tag: INSDC first public
attributes.value: 2021-05-26T00:29:51Z
attributes.tag: INSDC last update
attributes.value: 2021-05-26T00:29:51Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9331:G225-3_USP22_KO
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: disease
attributes.value: HER2 positive breast carcinoma
attributes.tag: genotype
attributes.value: MMTV-Erbb2; MMTV-cre; USP22 fl/fl
attributes.tag: organism part
attributes.value: mammary gland
attributes.tag: replicate
attributes.value: 4
attributes.tag: sample name
attributes.value: E-MTAB-9331:G225-3_USP22_KO
attributes.tag: sampling site
attributes.value: neoplasm
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: FVB/N