title: GSM8543269: HeLa-S3, 240min, G1, Cytosol; Homo sapiens; OTHER
library_construction_protocol: At each time point, 3 million cells were fractionated to obtain nuclear and cytosolic fractions based on Mayer et al. (2015) with the following modifications: Nuclear pellets were washed with nuclear wash buffer (PBS with 1X cOmplete Protease Inhibitor (Roche, 11873580001)) and resuspended in 1400 µl TRIzol (Invitrogen, 15596026). Similarly, a time series in the G1 cell cycle phase (12 hours release time after double thymidine blocking) was measured at 0, 15, 45, 75, 145, 180, 240 and 300 minutes. Prior to phenol extraction of RNA, 1.9 µl of 1:100 diluted ERCC ExFold RNA Spike-In Mixes (Invitrogen, 4456739) per 1 million cells was added to each sample. The 4sU RNA was alkylated according to Herzog et al. (2017), and poly-A enriched SLAM-seq libraries were generated using the 3'-QuantSeq-REV protocol (Lexogen).