3p full length 2 rep 3
RNY5 mutant pools were transfected into NIH 3T3 murine cells using Fugene 6. After 24 h the cells were harvested and total RNA extracted. For generation of the full length libraries 5 l of total RNA was treated with Turbo DNase for 30 min at 37C. Reverse transcription was carried out using the Superscript II protocol according to the manufacturers instructions. The Illumina adapter and index sequences were introduced by PCR. The PCR product was purified and quantified. The cloned cDNA libraries were sequenced on a HiSeq 2500 with 50 bp single end sequencing at the Earlham Institute (Norwich Research Park).