N-terminal tagging of RNA Polymerase II shapes transcriptomes more than C-terminal alterations

To investigate the effect of RPB1 CTD length on transcription dynamics, six mutant U2OS cell lines expressing tagged versions of a-amanitin resistant RPB1 with CTDs of varying lengths (Dendra2-RPB1-25R (D25), Dendra2-RPB1-52R (D52), Dendra2-RPB1-70R (D70), HaloTag-RPB1-25R (H25), HaloTag-RPB1-52R (H52), and HaloTag-RPB1-70R (H70)) were grown in a-amanitin to degrade endogenous RPB1, then their RNA was sequenced at single-cell resolution using a droplet-based system. Overall design: The three HaloTag cell lines (H25, H52, and H70) were sequenced on 25-FEB-2021 but the target number of reads was not obtained, so the same libraries were sequenced again on 23-MAR-2021. Separate sequencing runs of the same library were treated as one sample when processing. The three Dendra2 cell lines (D25, D52, and D70) were sequenced on 16-JUN-2021.