SH-SY5Y_Exon9Exon11_DMStreated
Approximately 30 million SHSY-5Y cells were harvested by centrifugation and resuspended in bicine buffered medium (300 mM Bicine pH 8.3, 150 mM NaCl, 5 mM MgCl2) followed by treatment with DMS (1:10 ethanol diluted) for 5 min at 37C. After incubation, the reactions were neutralized by the addition of 200 l of 20% by volume -mercaptoethanol. Total RNA was extracted by Trizol (ThermoFisher Scientific), treated with TURBODNase (ThermoFisher Scientific), 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: GCAGTGGTCCGTACTCCAC (fwd), TGATGGATGTTGCCTAATGAGC (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).