GSM5648166: SPAR-seq of 108 multi-exon gene regions in N2A cells, Xab2 knockdown (T1507), replicate 1; Mus musculus; OTHER
Forty-eight hours post-transfection, total RNA were purified from cultured cells using RNeasy Plus 96 Kit (Qiagen), as per the manufacturer's instructions. For each sample, a multiplex RT-PCR assay was first applied to simultaneously amplify 108 AS and/or expression events in a single reaction. The multiplex RT-PCR reaction was carried out in 96-well plates using the OneStep RT-PCR kit (Qiagen) as recommended by the manufacturer, with the following changes: reactions were performed in a volume of 20 uL with 2 uL of the purified total RNA as input, and a mixture of 50 pairs of primers was added to each reaction with a final concentration of 0.25 uM for each individual forward and reverse primer. Four identical Veriti 96-well Thermal Cyclers (Applied Biosystems) were used with the following conditions: 50°C for 30 minutes, 95°C for 15 minute, 30 cycles of 94°C for 40 seconds, 58°