Illumina HiSeq 2500 sequencing; 3' UTR length and mRNP composition determine endocleavage efficiencies at termination codons
3' UTR length and mRNP composition determine endocleavage efficiencies at termination codons
TPI_PTC189_1_CCGG
Standard lab conditions were used to grow the HeLa cells used for these experiments HeLa cells were grown over night in 6 cm plates and transiently transfected with 300 pmol siRNA using Lipofectamine RNAiMAX (Life Technologies). 24 h post transfection the cells were split 1:2 in 10 cm plates and the day after transfected again with 600 pmol siRNA. RNA samples were obtained from XRN1 knockdown HeLa cells transfected with the indicated reporter mRNA expressing plasmids (TPI-WT or -PTCs). Total RNA was extracted using Isol-RNA-Lysis Reagent. 3' fragments were linker ligated, reverse transcribed and specific PCR products were amplified with gene specific primers.