GSM7732400: Py8119_Ribotag_Tumor_63A6_1_Input; Mus musculus; RNA-Seq
After the mice were euthanized, tumors were rapidly resected and snap frozen in liquid nitrogen. Samples were stored at -80oC until polysome immunoprecipitation. To isolate actively translating mRNA from endothelial cell polysomes, the tumor tissue was pulverized with a mortar and pestle in liquid nitrogen and suspended in RNA tissue lysis buffer [50mM Tris pH 7.5 (Invitrogen), 100mM KCl (Invitrogen), 15mM MgCl2 (Invitrogen), 100μg of  cycloheximide (Sigma Aldrich), 1% Triton X-100 (Fisher Sci), 1mM DTT (Sigma Aldrich), 7.5μL/mL Protector RNA (Roche), 12μL/mL Turbo DNAse (Thermofisher), 10μL/mL Halt Protease Inhbitor (Thermofisher), RNAse-Free H2O (Fisher Sci)]. The mixture was transferred to a glass Dounce homogenizer on ice and then lysed with 10 strokes of pestle A followed by an additional 10 strokes with pestle B. The lysed mixture was transferred to an ice-cold microcentrifuge tube and spun at 16000x G for 10mins at 4oC. The supernatant was aspirated and 100μL was set aside for input analysis. The remainder of the supernatant was added to Dynabeads (Thermofisher) conjugated to anti-HA antibodies (Abcam) and rocked on ice for 2hrs. The beads were separated by magnetic separation, washed twice with wash buffer, and then eluted using TRIzol (Thermofisher). The mRNA was isolated using a TRIzol Microprep Kit (Zymo Research) and eluted into 13μL of RNAse free H2O. Prior to submitting the samples for sequencing, RNA quality and concentration was assessed using an Agilent TapeStation 4200. Sequencing was performed on the NovaSeq 6000 (SP flow cell, 100bp single reads) RNA quality and concentration was assessed using an Agilent TapeStation 4200. Sequencing was performed on the NovaSeq 6000 (SP flow cell, 100bp single reads)