Day 3 5' RACE of L1TfI/II on PacBio Sequel II Replicate 1
Total RNA was extracted from E14Tg2a mESCs differentiated to embryoid bodies as described above, at day 0, day 3, and day 21 of the differentiation protocol using the Qiagen RNasy Mini kit (cat. no. 74104). 5RACE was performed using the 5RACE module of the Takara SMARTer 5/3 RACE Kit (cat. no. 634858) according to the manufacturers protocol. 1 g total RNA was used as input for each reaction, and two reactions from biologically independent samples were performed per time point. PCR amplification of 5RACE cDNAs was performed using the L1 gene specific primer 5- catctcttgtattctgttgctgatgctcaa-3 and the 5RACE universal primer provided in the Takara SMARTer 5/3 RACE Kit. 25 PCR cycles were performed as follows: 94C for 30 sec, 67 C for 30 sec, 72 C for 3 minutes. PCR products were visualised on a 1% agarose gel. The resultant smear was excised, and PCR fragments were purified using the Qiagen MinElute Gel Extraction Kit (cat. no. 28604). Iso-Seq template preparation using the Iso-Seq Express kit followed by PacBio SMRT Cell sequencing on a PacBio Sequel II platform was performed by the Australian Genome Research Facility. Six samples (two replicates per time points d0, d3, and d21) were multiplexed on a single SMRT cell,