GSM8279918: HEK293T EXOSC3 siRNA PAS-seq Replicate 2; Homo sapiens; OTHER
Total RNA was extracted from Trizol following the manufacturer's protocol PAS-seq libraries were prepared as previously described (PMID: 34183125),  with the following minimal modifications. Purified mRNA was fragmented at 94˚C for 3 minutes using the NEBNext Magnesium RNA fragmentation module. In addition, the final libraries were run out on a 2.5% standard agarose gel and the region between 200-300 basepairs was gel extracted and sequenced. For all samples, a small amount of mouse RNA was spiked in but was not used in data processing. For the spike-ins, an extra control or treatment well was prepared and the cells in this extra well were counted and recorded. 1 µg total RNA from each sample was used and up to 100 ng (10%) spike-in RNA purified from mouse brain tissue was added based on the original cell number. For example, if there were 1 x 10^6 control cells and 0.8 x 10^6 treated cells, then 100 ng mouse spike-in RNA would be added to 1 µg of control total RNA and 80 ng mouse spike-in RNA would be added to 1 µg of treated total RNA. As such, the level of spike-in RNA could be used to normalize sequencing reads to the original cell number.