title: T47D_Exon10Intron10_Exvivo_DMStreated
design: Approximately 20 million T47D cells were harvested by centrifugation and resuspended in bicine buffered medium (300 mM Bicine pH 8.3, 150 mM NaCl, 5 mM MgCl2). Total RNA was extracted by Trizol (ThermoFisher Scientific) followed by treatment with DMS (1:10 ethanol diluted) for 5 min at 37C. For the negative control (unmodified RNA), instead of DMS, an equivalent amount of ethanol was added to cells. After incubation, the reactions were neutralized by the addition of 200 l of 20% by volume -mercaptoethanol. RNA was purified using Purelink RNA mini kit (ThermoFisher Scientific), and quantified with NanoDrop spectrophotometer. Purified RNA (9 g) was reverse-transcribed using Random Primer 9 (NEB) and SuperScript II reverse transcriptase under error-prone conditions as described in 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). The following PCR primers used for library generation: AACGTCCAGTCCAAGTGTGG (fwd), CATTCACCCAGAGGTCGCAG (rev). The cDNA was amplified by the NEB Q5 HotStart polymerase (NEB). Secondary PCR was performed to introduce TrueSeq barcodes (Smola et al. 2015). All samples were purified using the 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. TrueSeq libraries were then sequenced as necessary for their desired length as 2301 read multiplex runs on MiSeq platform (Illumina).