RNA-Seq polysome profiling in OCCL treated with the MAP3K8 kinase inhibitor

Sucrose density gradient centrifugation was performed to separate the subpolysomal and polysomal ribosome fractions. For polysome profiling 30x10^6 IGROV1 at 70% confluence were needed after treatment with 10 µM MAP3K8-ki or DMSO for 1 hour before 10% FBS stimulation for 2 h. For cytoplasmic lysate preparation, 15 minutes prior to collection, the cells were incubated at 37 °C with 100 µg/ml cycloheximide, then washed, scraped into ice-cold PBS supplemented with 100 mg/ml cycloheximide, centrifuged at 3,000 r.p.m. for 5 min and resuspended into 400 µl of LSB buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 3 mM MgCl2, 0.5 M sucrose, 2.4% Triton X-100, 1 mM DTT, 100 U/ml RNasin and 100 µg/ml cycloheximide). After homogenization, 400 µl of LSB buffer supplemented with 0.2% Triton X-100 and 0.25 M sucrose were added. The samples were centrifuged at 12,000g for 10 min at 4 °C. The resulting supernatant was adjusted with 5 M NaCl and 1 M MgCl2. The lysates were loaded onto a 15–50% sucrose density gradient and centrifuged in an SW41 rotor at 38,000 r.p.m. for 2 h at 4 °C. The polysomal fractions were monitored and collected using a gradient fractionation system (Isco). Three independent experiments were performed, and total RNA was extracted from 75µl of the four heaviest fractions pooled and input samples using the TRIzolTM -LS (ThermoFisher Scientific #10296010) according to the manufacturer’s instructions for polysome-bound mRNA-sequencing (polysome-bound mRNA-seq) and total mRNA-sequencing (mRNA-seq), respectively. RNA integrity and quality were analyzed with the Agilent RNA 6000 Pico kit. RNA sequencing libraries were prepared from total RNA prep using the Illumina® Stranded mRNA Prep Ligation library preparation kit, which allows to perform a strand specific sequencing. This protocol includes a first step of polyA selection using magnetic beads to focus sequencing on polyadenylated transcripts. After RNA fragmentation, cDNA synthesis was then performed and resulting fragments were used for dA-tailing followed by ligation of RNA Index Anchors. PCR amplification with indexed primers (IDT for Illumina RNA UD Indexes) was finally achieved, with 15 cycles, to generate the final cDNA libraries. Individual library quantification and quality assessment was performed using Qubit fluorometric assay (Invitrogen) with dsDNA HS (High Sensitivity) Assay Kit and LabChip GX Touch using a High Sensitivity DNA chip (Perkin Elmer). Libraries were then equimolarly pooled and quantified by qPCR using the KAPA library quantification kit (Roche). Sequencing was carried out on the NovaSeq 6000 instrument from Illumina using paired-end 2 x 100 bp, to obtain around 30 million clusters (60 million raw paired-end reads) per sample.

• 27/07/2026
• 28 samples
• DAC: EGAC00001000581
• Technology: Illumina NovaSeq 6000

• Archive: European Genome-phenome Archive (EGA)