BioSample

Total RNA was extracted using Trizol (Invitrogen) and messenger RNA was isolated using Nucleotrap mRNA kit (Macherey-Nagel). First strand cDNA was prepared using the Creator SMART cDNA Library Construction Kit (Clontech) with two modifications: a modified 3' primer (5'-AAGCAGTGGTATCAACGCAGAGTGGCCGAGGCGGCCTGTTTTGTTTTTTTTTCTTTTTTTTTTVN-3') and a 1:1 mixture of ArrayScript Reverse Transcriptase (Ambion) and BioScript Reverser transcriptase (Bioline). Second strand cDNA was synthesised using the Advantage 2 PCR System (Clontech) with adapter-specific primer (5'-AAGCAGTGGTATCAACGCAGAGT-3'). Double stranded cDNA was purified using the DNA Clean And Concentrator (Zymogen) and digested with Sfi-I restriction enzyme (New England Biolabs). Digested cDNA was purified and ligated to pDNR-Lib vector (Clontec) and heat transformed into JM109 (Promega). 576 random colonies were picked and the cDNA inserts were amplified using M13 primers. Unincorporated nucleotides and oligos were removed using ExoZap (Stratagene) and the amplicons were sequenced from the M13F end.