3' sequencing of nuclear and cytoplasmic fractions following treatment with a CLK inhibitor
3' sequencing of nuclear and cytoplasmic fractions following treatment with a CLK inhibitor
Samples were collected by trypsinising, spinning down, washing in PBS, spinning down and snap freezing on dry ice. Cells were grown in DMEM+Glutamax+10% FBS and seeded such that they achieved 70% confluency on the morning of the experiment, with each sample being derived from 2 wells of a 6 well plate. Samples were treated for 8 hours with either 1 uM CLK-IN-T3 or the equivalent volume of DMSO. RNA extractions were performed from cell pellets using the Maxwell RSC simplyRNA kit (Promega) using a Maxwell RSC Instrument (Promega). RNA was fragmented using magnesium ions and heat, purified, and reverse transcribed using an oligo-dT primer with an Illumina P7 sequence and UMIs. A template switching oligo with the P5 sequence was included in the reaction. The library was then amplified with indexed Illumina i5/i7 primers.