title: Illumina HiSeq 4000 sequencing; RNA-seq on HER2-driven murine mammary carcinomas (MMTV-Erbb2; MMTV-cre) without or with a cre-mediated homozygous deletion of Usp22 (MMTV-Erbb2; MMTV-cre; Usp22 fl/fl)
design: RNA-seq on HER2-driven murine mammary carcinomas (MMTV-Erbb2; MMTV-cre) without or with a cre-mediated homozygous deletion of Usp22 (MMTV-Erbb2; MMTV-cre; Usp22 fl/fl)
library_construction_protocol: 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: Experimental Factor: genotype
attributes.value: MMTV-Erbb2; MMTV-cre