title: GSM5026398: ShFTO-Light-3; Homo sapiens; OTHER
library_construction_protocol: Transcriptome Analysis. Total RNA was extracted using TRIzol method. Polysome fractionation. 6 plates (150 cm2) were seeded with 2 x 106 cells. After 72 h, cells were treated with 20 µg / ml emetine for 5 min at 37°C, washed twice with ice-cold PBS, and scraped in ice cold PBS. Cells were centrifuged, resuspended into 1mL of polysome lysis buffer, homogenized by hard shaking with 1,4mm ceramix spheres (Lysing matrix D MPBio) in FastPrep machine (MPBio), centrifuges 10 min at full speed at 4°C. Lysates were loaded on 15-50% sucrose gradient and centrifuged at 35,000 rpm for 2.5 h at 4°C in a SW41 rotor (Beckman Coulter). Polysomes were separated through a live optical density (OD) 254 nm UV spectrometer and collected with an ISCO (Lincoln, NE) density gradient fractionation system. The absorbance at 254 nm was measured continuously as a function of gradient depth. Translatome Analysis. mRNA associated with 1 to 3 ribosomes “Light polysomes”  and mRNA associated with more than 3 ribosomes “Heavy polysomes” were extracted using TRIzol LS (Invitrogen) according to manufacturer's instructions. Merip Analysis. 5 µg of mRNA were fragmented (Invitrogen AM8740) by adding 1 µL of 10X fragmentation reagent to 9 µL mRNA and incubated at 95˚C for 1 min. The same samples were then pooled prior to overnight EtOH precipitation (0.1 volume of 3M sodium acetate pH5.2, 2.5 volume of pure EtOH, 1 µL glycogen). The fragmented mRNA were recovered by centrifugation at 14,000rpm 25 min at 4˚C and the pellets were washed once with 70% EtOH and centrifuged again 14,000rpm 10 min at 4 ˚C. The pellets were resuspended in 429 µL DEPC water and 390 µL was used for m6A-methylated RNA immunoprecipitation and 39 µL for inputs (10%). Transcriptome Analysis. RNA samples were send to Eurofins Genomics /GATC to perform next generation sequencing on Illumina platform (2 x 150bp). Library preparation was performed using TruSeq Stranded mRNA Sample Preparation kit (Illumina) according to the manufacturer's protocol. Translatome Analysis.  Library preparation was performed using TruSeq Stranded mRNA Sample Preparation kit (Illumina) according to the manufacturer's protocol. cDNA libraries were sequenced using the sequencer HiSeq 2500 (Illumina). Merip Analysis. Multiplex cDNA Library was produced using NEBNext Small RNA Library Prep Set for Illumina (NE BiolabsInc E7330S), according to manufacturer procedure. Sequencing was performed using NextSeq 500.