Transcript 5′ termini were determined employing a 5′-RACE technique described by Bensing et al. (27 (link)) with the following modifications. 5′ triphosphates were converted to monophosphates by treating 5 μg RNA with 10 U of tobacco acid pyrophosphatase (TAP) (Epicentre Technologies) at 37°C for 1 h in the presence of 40 U of RNase inhibitor (Fermentas GmbH, Germany) in the appropriate buffer. Control reactions were set up without pyrophosphatase. The RNA was subsequently extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated from the aqueous phase by adding 3 vol of ethanol/3 M sodium acetate, pH 5.2 (30:1) and dissolved in water. The RNA was then supplemented with 10 pmol 5′ RNA adapter A3 (28 (link)), and the ligation of transcripts to the adapter was performed at 37°C for 1 h with 50 U of T4 RNA ligase (Epicentre Technologies) in the presence of 1 mM ATP and 80 U of RNase inhibitor (Fermentas GmbH) in the appropriate buffer. Control reactions were set up without adding the adapter. Following the ligation, the RNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated by adding 3 vol of ethanol/3 M sodium acetate, pH 5.2 (30:1), dissolved in water and then reverse-transcribed using gene-specific primers and Omniscript Reverse Transcriptase (Qiagen, Germany) according to the manufacturer's protocol. The products of reverse transcription were amplified in a first PCR step by using 1–3 μl of the RT reaction, 5 pmol of each adapter-specific forward primer P1a (5′-CGA ATT CCT GTA GAA CGA ACA CTA GAA G-3′) and gene-specific reverse primer, 200 μM of each dNTP and 0.5 U of Taq DNA polymerase (Qiagen) in 25 μl of the appropriate buffer. Cycling conditions: 94°C for 1 min; 35 cycles of 95°C for 20 s, 58–62°C for 20 s, 72°C for 2 min; 72°C for 10 min. An aliquot of 0.1–1 μl of the first PCR reaction was used as template for subsequent nested PCRs set up essentially as the first PCR in a volume of 50 μl with 10 pmol of each gene-specific and adapter-specific primer. Gene-specific primers were repeatedly placed upstream of identified transcriptional starts, until no 5′-RACE products reaching further upstream could be detected. PCR reactions were analysed on gels composed of 1% agarose and 2% Nusieve agarose (Biozym, Germany). Products of interest were excised, purified over QIAquick spin columns (Qiagen) and ligated into pDrive (Qiagen). Ligation products were transformed into Escherichia coli TOP10 (Invitrogen, Germany). Bacterial clones containing the plasmid insert were identified by colony PCR with vector-specific primers. Colony PCR reactions were set up and performed essentially as the first PCR of the 5′-RACE protocol, and PCR products were purified over QIAquick spin columns (Qiagen) and sequenced employing an ABI 377 automatic DNA Sequencer (Applied Biosystems).
A list of all gene-specific primers used in 5′-RACE reactions is provided in the Supplementary Material.