GSM4432366: SF3A3 5'UTR WT in vitro DMS-MaPseq; synthetic construct; OTHER
Cells were collected by centrifugation and total RNA extracted with TRIzol Reagent (ThermoFisher Scientific, #15596018). After phase separation, the aqueous phase was added to 1 ml of 100% ethanol and the RNA purified on RNA Clean & Concentrator-5 columns (Zymo Research, #R1016). For reverse transcription, 2 ug of total RNA were heated at 70°C for 5 min, in the presence of 1 uM final gene specific primer and 1 mM final each dNTP. Sample was then immediately transferred to ice for 1 min, after which reaction was supplemented with RT buffer [50 mM Tris pH 8.3; 75 mM KCl; 3 mM MgCl2], 5 mM final DTT and 10 U SuperaseIN RNase Inhibitor (Invitrogen, #AM2694), and incubated at r.t. for 5 min. 100 U of TGIRT-III RT enzyme (InGex) were then added and the reaction was allowed to proceed at 50°C for 5 min, followed by 2 h at 57°C. The enzyme-cDNA-RNA complex was resolved by addition of 1 ul NaOH (5 N) followed by 3 min at 95°C. cDNA was then purified using RNA Clean & Concentrator-5 columns and 20% of cDNA was used for PCR using gene-specific primers and TaKaRa Taq™ DNA Polymerase (TaKaRa, #R0001A). To prepare libraries for the endogenous WT SF3A3 5' UTR, the PCR amplicon was first sheared into fragments of ~150-200 bp. Library preparation was performed using 10 ng of sheared DNA and the NEBNext Ultra II DNA Library Prep kit (NEB, #E7645S), following manufacturer instructions. To prepare libraries for the Fluc constructs, gene-specific primers were fused with Illumina adaptor overhangs. After a first round of PCR, library barcodes were introduced by a second round of PCR. Libraries were then sequenced on a Illumina NextSeq 500 instrument.