GSM3389674: H358-OXPT-T-2; Homo sapiens; RNA-Seq
RNA was extracted from total cells, cytosol fraction or heavy polysomes. 5 μg of total RNA were used for poly(A)+ RNA purification with Dynabeads mRNA DIRECT Micro kit (ThermoFisher Scientific). Poly(A)+ RNA was fragmented at 70°C for 5 min in RNA Fragmentation Reagents (ThermoFisher Scientific). After ethanol precipitation, RNA was reverse transcribed using anchored oligo-dT and SMARTScribe RT enzyme (Clontech) allowing to add adapter sequences to the 5' and 3' ends of RNA fragments. Libraries were amplified by 16 cycles of PCR with GoTaq Flexi DNA Polymerase (Promega), purified using Agencourt AMPure XP (Beckman Coulter) magnetic beads, and quantified with Quant-iT Picogreen dsDNA kit (ThermoFisher Scientific). A size selection step was done using SPRIselect (Beckman Coulter) magnetic beads to obtain fragments of 150–300 bp. Purified libraries were controlled by capillary electrophoresis (LabChip - Perkin) and quantitated by qPCR (KAPA Library Quantification Kits Illumina Platforms - Roche). Pooled libraries (12 pM) with 30% of phiX were subjected to single-end, 50 bp sequencing using the HiSeq 2500 machine (Illumina). Read 1 was read with primer HP6 (Illumina) with 3 dark cycles (first 3 bases of read 1 were not read). Index i7 (6 pb barcode) was read with primer HP8 (Illumina).