GSM9230577: RPB1degron_62nM_dTAGv1_1; Homo sapiens; OTHER
PRO-seq was performed as described previously. Briefly, DMSO or dTAGV-1-treated cells or Drosophila S2 cells were permeabilized and counted. 1 million permeabilized cells (nuclei) per sample were mixed with 5% spike-in Drosophila S2 permeabilized cells (nuclei) in 50 μl, then mixed with 50 μl 2X Run-On Master Mix containing biotin-tagged CTP and UTPs (PerkinElmer, NEL542001EA-NEL545001EA) to perform the run-on reaction at 37°C for 5 min. The reaction was terminated by adding TRIzol LS followed by total RNA extraction. Total RNA was denatured by adding NaOH to 200 mM and then purified on a Bio-Rad RNase free P-30 column following the manufacturer's instructions. Then the 3' RNA adaptor ligation was performed, and the biotin labeled RNA was enriched on Streptavidin beads. Next 5' hydroxyl repair and de-capping reactions were performed on-bead followed by 5' RNA adaptor ligation. After these enzymatic reactions, the Streptavidin beads with bound biotin labeled RNA were washed. Biotin labeled RNA was released from the Streptavidin beads using TRIzol/ chloroform extraction followed by precipitation. Reverse transcription was performed off-bead to get cDNA. 1.54 μl out of the 20 μl reverse transcription reaction was used to do a test amplification (qRT-PCR) to determine the amplification cycle number, then full-scale amplification was performed with 15 cycles. PCR products were cleaned up and selected on SPRI beads for sequencing. The libraries were then sequenced on an Illumina NextSeq 2000 (Genomics Platform at University of Copenhagen), yielding 100bp single-end reads.