organism: Mus musculus
description: Protocols: Tumors were excised from mice just after the mice were killed, and put into a cryo-tube and snap frozen in liquid nitrogen RNA was extracted using the Trizol-Chloroform-Isopropanol method: Frozen tumors were manually homogenized to powder using mortar and pestle cooled on dry ice. Tissue, a metal bullet and 1 ml Trizol was added to cool 2 ml eppendorf tubes, and tissue was homogenized in a Qiagen Tissuelyser at 25 Hz for 2-3 x 1,5 min, and incubated 5 min at RT. 200 ul Chloroform was added to all samples and shaken. Samples were incubated for 3-5 min at RT to ensure dissociation of nucleic acids from protein complexes, and spun at 12,000 g for 15 min, 4 ºC. The upper clear aqueous phases (approx. 450 ul) were transferred to new PCR clean tubes. This is the RNA. RNA was precipitated with 0.45 ml isopropyl alcohol (1:1 reaction) at RT, and samples were vortexed for 5-10 sec and incubated 10 min at RT. Samples were spun at 12000 g, 15 min, 4 ºC, and placed in -20C cold blocks. The aqueous phases were poured out, and pellets were washed with 900uL (-20C chilled) 75% ethanol and spun at 8000 g, 5 min, 4ºC. This step was repeated once. Pellets were dried by placing tubes horizontally on a rack so that the tops “hung off” the edge of the rack and the air flow from the hood moved past the open tops of the tubes in a perpendicular manner. Tubes were left for 3-5 minutes until pellets had “just” dried and had NO remaining liquid. Pellets were dissolved in 15-30µl of Ultrapure RNase/DNase free water. Sequencing libraries were prepared from 1μg total RNA using the TruSeq stranded mRNA library preparation kit (Cat# RS- 122-2101/2102, Illumina Inc.) including polyA selection. The library preparation was performed according to the manufacturers' protocol (#15031047). Library preparation using TruSeq stranded mRNA sample preparation kit is an accredited method.
attributes.tag: ENA first public
attributes.value: 2019-08-14
attributes.tag: ENA last update
attributes.value: 2017-01-05
attributes.tag: External Id
attributes.value: SAMEA39850168
attributes.tag: INSDC center alias
attributes.value: CHALMERS
attributes.tag: INSDC center name
attributes.value: Chalmers University of Technology
attributes.tag: INSDC first public
attributes.value: 2019-08-14T17:03:45Z
attributes.tag: INSDC last update
attributes.value: 2017-01-05T15:31:10Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5311:Sample 10
attributes.tag: age
attributes.value: 24 to 26
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: disease
attributes.value: Lewis lung carcinoma
attributes.tag: environmental history
attributes.value: access to running wheel
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: sample name
attributes.value: E-MTAB-5311:Sample 10
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: C57BL/6