Calf alkaline phosphatase

TSS were identified using the RLM-RACE (Ambion) kit followed by cloning 5’rapid amplification of cDNA ends (5’RACE) products into the pCR4-TOPO vector. Briefly, DNase I- treated RNA was phenol:chloroform:isoamyl alcohol (25:24:1, Invitrogen) extracted and treated with calf alkaline phosphatase (Ambion). Reverse transcription of the RNA adapter-tagged sample was primed using gene specific primers (sequences in Supplemental Table S1) using Superscipt III (Invitrogen ) at 50 °C, following manufacturer instructions. A first PCR was carried as described above for 32 cycles using the “outer” Ambion primer with the 1^st nested gene-specific primer (Supplemental Table I). A second (nested) PCR was carried out as above using the “inner” Ambion primer with the 2^nd nested gene-specific primer, at one of several annealing/extension temperatures (54 – 68 °C, 60 s) for 40 cycles. Products were purified using a Qiagen spin column and cloned into the pCR4-TOPO vector using the TOPO-TA cloning kit (Invitrogen). Sequencing was carried out by the Northwestern University sequencing facility (Chicago, IL).