GSM7814511: RNA_Proargdrop_DFMO_5732; Mus musculus; RNA-Seq
Mouse tumors were mechanically disrupted in liquid nitrogen and homogenized in a lysis buffer (LB, 50 mM Tris, pH 7.4, 100 mM KCl, 1.5 mM MgCl2, 1.0% Triton X-100, 0.5% Na-Deoxycholate, 25 U/ml Turbo DNase I, 1mM DTT, 100 µg/ml cycloheximide, and Protease inhibitors) 3 ml of LB per 1 g of tissue. Ribosome profiling: Mouse tumors were mechanically disrupted in liquid nitrogen and homogenized in a lysis buffer (LB, 50 mM Tris, pH 7.4, 100 mM KCl, 1.5 mM MgCl2, 1.0% Triton X-100, 0.5% Na-Deoxycholate, 25 U/ml Turbo DNase I, 1mM DTT, 100 µg/ml cycloheximide, and Protease inhibitors) 3 ml of LB per 1 g of tissue. To obtain ribosome footprints 0.12 ml of total extracts containing 300 µg of total RNA were treated with RNAse I (Epicentre) (25U/1 mg of total RNA), for 45 min at 20ºC with slow agitation. 10 ml SUPERaseIn RNase inhibitor was added to stop nuclease digestion. Monosomes were isolated using S-400 columns. For isolation of mRNA protected fragments (RPF) 3 volumes of QIAzol® were added to the S-400 eluate, mixed thoroughly and proceed to RNA purification with Direct-Zol RNA Mini Prep Plus kit. RNA-seq: Total RNA were isolated from the same extracts, that were used to obtain RPF (bellow, RiboSeq). 3 volumes of QIAzol® (Qiagen, Cat. No. 79306) were added to 80 µl of cell extracts, mixed thoroughly and proceed to RNA purification with Direct-Zol RNA Mini Prep Plus kit. Ribosome Profiling: RPF libraries were prepared as described (PMID: 28579404, PMID: 19213877). Briefly, RPFs (25-34 nt) were size-selected by electrophoresis using a 15% TBE-Urea polyacrylamide gel electrophoresis (PAGE) and two RNA markers, 25-mer (5' AUGUACACGGAGUCGAGCACCCGCA 3') and 34-mer (5'AUGUACACGGAGUCGAGCACCCGCAACGCGAAUG 3'). After dephosphorylation with T4 Polynucleotide Kinase (NEB, #M0201S) the adapter Linker-1 (5' rAppCTGTAGGCACCATCAAT/3ddC/ 3') was ligated to the 3′ end of the RPF using T4 RNA Ligase 2. Ligated products were purified using 10% TBE-Urea PAGE. Ribosomal RNA was subtracted using RiboCop rRNA Depletion Kit V2 H/M/R. The adapter Linker-1 was used for priming reverse transcription (RT) with the RT primer Ni-Ni-9 (5'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC5CACTCA5TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG 3') using ProtoScript® II Reverse Transcriptase. RT products were purified using 10% TBE-Urea PAGE. The cDNA was circularized with CircLigase™ II ssDNA Ligase. The final libraries were generated by PCR using forward index primer NI-N-2 (5' AATGATACGGCGACCACCGAGATCTACAC 3') and one of the reverse index primers. Amplified libraries were purified using 8% TBE-PAGE and analyzed by Qubit and TapeStation. RNAseq: RNA were sent to Genomic Platform (UNIGE) for stranded mRNA libraries preparation. Libraries were sequenced on an Illumina NovaSeq 6000, SR 100 bp, 10 libraries in 1 pool.