Targeted sequencing of HCT116 cell line: total RNA treated with Nutlin
HCT116 cells were cultured in RPMI (Gibco, Life Technologies, Milan, Italy) supplemented with 10% FBS, antibiotics (100 units/ml penicillin plus 100 mg/ml streptomycin), and 2mM L-glutamine. Polysomal profiling was performed in DMSO control or after 10M Nutlin treatment (10M, 16 hours). Cytoplasmic lysates were prepared, loaded in 1550% linear sucrose gradients, ultra-centrifuged in a Beckman SW41 rotor at 40.000 rpm for 1 hour and 40 minutes at 4C, and fractionated with a Teledyne Isco model 160 gradient analyzer equipped with a UA-6 UV/VIS detector. All fractions or polysome-associated mRNAs were identified from the absorbance profile and pooled into 15 ml falcon tubes. Total cytoplasmic RNA was reconstituted using an aliquot from each individual fraction. RNA was purified with the TRIzol reagent (Invitrogen TM ), following the manufacturers protocol. Targeted RNA-seq libraries were prepared using custom multiplex primers, each containing an ~25- nucleotide (25-nt) gene-specific sequence and either a 33-nt Illumina adaptor (5- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3) at the 5 end of the forward primer or a 34-nt Illumina adaptor (5-GTCTCGTGGGCTCGGAGATGT GTATAAGAGACAG-3) at the 5 end of the reverse primer. Targeted RNA-seq libraries were prepared from DNase-treated RNA. cDNA was synthesized using a dTVN primer and purified with SPRIselect beads to remove excess primers. Fifty nanograms (50 ng) of cDNA was amplified (first PCR) using gene-specific forward and reverse primers attached to Illumina-specific adapters for cluster generation and sequencing (5-Illumina Nextera adaptor + sequence-specific forward and reverse primer sequence-3). A five-cycle second PCR was performed using Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific, Waltham, MA) to incorporate Illumina Nextera primers for sample indexing. After purification with Beckman Ampure XP beads, quantification with Qubit, and quality control using the PerkinElmer LabChip GX, the indexed libraries were pooled, quantified by qPCR using the Roche KAPA Library Quantification Kit (for Illumina platforms), and sequenced on the Illumina MiSeq platform (Illumina, San Diego.