title: RT-PCR of Lariat Amplicon: HeLa, SF3B1 K700E, DPH5, rep2
design: To extract RNA from cell lines, HeLa cells of SF3B1 wildtype or K700E background  were grown to 80-90% confluence in DMEM+10% FBS supplemented with 1000 U/ml of each penicillin and streptomycin (Gibco, Cat#15140-122) at 37 °C in 5% CO2. Total RNA was isolated using Trizol reagent according to the manufacturer’s instructions and suspended in DEPC water. 1 g of the samples were digested with Amplification grade DNase I (Invitrogen, Cat#18068) according to the manufacturer’s instructions to remove any DNA contamination. cDNA was synthesized using random 9-mer primers (Integrated DNA Technologies) and SuperScript III Reverse Transcriptase (Invitrogen, Cat#18080) according to the manufacturer’s instructions. PCR was then performed on 1l of cDNA using the outer primer pair with GoTaq Green Master Mix (Promega, Cat#M7122) in a total volume of 20l according to the manufacturer’s instructions. A second PCR using “nested” primers was then performed with 0.5l of the initial PCR product used as the template. PCR was performed using Bio-Rad PCR machine programmed for 5 min at 95 °C for initial denaturation, followed by 35 cycles of 20 s at 95 °C for denaturation, 25 s at 55 °C for annealing and 25 s at 72 °C for extension. The final extension was set for 5 min at 72 °C. The product of the second PCR was then examined by electrophoresis at 150 V for 30 min in a 2% agarose gel in 1 x TAE buffer. The appropriate bands, if required, were excised and purified using a Qiagen gel extraction kit (Qiagen, Cat#28704). PCR products were then cloned into pCR2.1 using a TOPO TA Cloning Kit (Invitrogen, Cat#K4500) and transformed into TOP10 E. coli cells. Individual colonies were grown in LB+ampicillin media, and plasmid DNA was isolated using a Qiagen Miniprep Kit (Qiagen, Cat#27106), and was subsequently sequenced using Illumina MiSeq.