T47D_40S_ribosome_EtOHCntrl
Approximately 10 million T47D cells in 10 cm plates were used for each condition. We removed the growth media, added 1.8 mL of bicine buffered growth medium (200 mM Bicine pH 8.3) followed by treatment at 37C with 200 uL of DMS diluted in ethanol (1.25% final DMS) for 5 min respectively. For the negative control (unmodified RNA), instead of DMS, an equivalent amount of ethanol was added to the same number of T47D cells. After incubation, all reactions were neutralized by addition of ice cold 20% by volume -mercaptoethanol and kept on ice for 5 minutes. Total RNA was extracted by Trizol (ThermoFisher Scientific), chloroform and isoamyl alcohol using phase lock heavy tubes (5PRIME Phase Lock Gel). RNA was purified using Purelink RNA mini kit (ThermoFisher Scientific), treated with TURBODNase (ThermoFisher Scientific) and quantified with NanoDrop spectrophotometer. Purified RNA (5 ug) was reverse transcribed using Random Primer 9 (NEB) and SuperScript II reverse transcriptase under error prone conditions as described Smola et al., 2015. The resultant cDNA was purified using G50 column (GE healthcare) and subjected to second strand synthesis (NEBNext Second Strand Synthesis Module). Standard Nextera DNA library protocol (Illumina) was used to fragment the cDNA and add sequencing barcodes. All samples were purified using Ampure XP (Beckman Coulter) beads and quantification of the libraries was performed with Qubit dsDNA HS Assay kit (ThermoFisher Scientific). Final libraries were run on Agilent Bioanalyzer for quality check. Libraries were sequenced as paired end 2151 read multiplex runs on MiSeq platform (Illumina).