Five million cells at 50% confluency were co-transfected using JetPEI reagent with 0.5 g pX335, 1.5 g of each of the sgRNAs expression vectors and 1.5 g of repair plasmid (eIF4A3) or 0.5 g of modified pX335 and 4.5 g of repair plasmid. For CRISPR applications.

CLIP-seq methods provide transcriptome-wide snapshots of RNA-protein interactions in live cells. Reverse transcriptases stopping at cross-linked nucleotides sign for RNA-protein binding sites. Reading through cross-linked positions results in false binding site assignments. In the 'monitored enhanced CLIP' (meCLIP) method, a barcoded biotinylated linker is ligated at the 5' end of cross-linked RNA fragments to purify RNA prior to the reverse transcription. cDNAs keeping the barcode sequence correspond to reverse transcription read-throughs. Read through occurs in unpredictable proportions, representing up to one fourth of total reads. Filtering out those reads strongly improves reliability and precision in protein binding site assignment.