title: The type and relative distribution of the PP isolated RNAs by RNA-sequencing analysis in HeLa cells and dynamic investigation upon transcription inhibition by ActD treatment
library_construction_protocol: The HeLa cells were washed with 5mL cold PBS for three times, followed by irradiation with 254-nm UV light at 0.25 J/cm2 on ice using a UV cross-linker. After adding 2mL cold PBS, the cells were harvested using a cell lifter and collected in a 1.5-mL RNase-free tube.For RNA harvested by TRIzol samples,cells were harvested using a cell lifter and collected in a 1.5-mL RNase-free tube directly.   HeLa cells were grown in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a 5% CO2 atmosphere. For the actinomycin D (ActD) treatment experiment, HeLa cells were treated with 5 μM ActD at 37°C in DMEM medium (10% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin) for 0, 2, 5 and 9 hours before collecting the cells. For the actinomycin D (ActD) treatment experiment, HeLa cells were treated with 5 μM ActD at 37°C in DMEM medium (10% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin) for 0, 2, 5 and 9 hours before collecting the cells. The RP-complexes from HeLa cells were captured by PP, proteinase K  was added to digest protein, the eluted RNAs were further purified by TRIzol following the manufacturer's instructions. For other samples, RNA was harvested using Trizol reagent.    RNA was used in each replicate to construct the cDNA library using a NEBNext Ultra RNA Library Prep Kit for Illumina following the manufacturer's instructions.