The 5ʹ rapid amplification of cDNA ends (RACE) was performed using a 5ʹ/3ʹ RACE kit (Roche, Germany), as previously described [10 (link)]. The DNase I-treated total RNA was reverse-transcribed using a specific primer BT4204 (SP1). The first-strand cDNA was purified prior to adding poly(A) to the 5ʹ-terminus using terminal transferase enzyme. The poly(A)-tailed cDNA was then PCR-amplified using a specific primer BT4617 (SP2) and an anchored oligo(dT) primer. The PCR product was cloned into pUC18, and the transcription start site (+1) was identified by DNA sequencing.
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