organism: Mus musculus
description: Protocols: Mouse ES cells were cultured on 0.2% gelatin-coated plates in ES medium (DMEM, supplemented with 15% FBS, LIF, 2-Mercaptoethanol, 2 mM L-Glutamine and 1x non-essential amino acids) at 37C and 5% CO2. Medium was changed daily and cells were split every second day. Polymerase run-on sequencing (qPRO-seq) was performed as previously described (cite original Kwak et al., 2013 and qPRO Judd et al., 2020) with some minor changes. For the experiment, 5 million cells were used per sample with a spike-in of 0.05 million (1%) Drosophila S2 cells. In contrast to the protocol, mouse ES cells were harvested using Trypsin-EDTA on ice and washed two times with cold PBS with centrifugations at 1000x g at 4C. Permeabilization was performed in 10ml permeabilization buffer (10 mM Tris-HCl pH 8.0, 250 mM sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.05% Tween-20, 0.1% NP40 substitute, 0.5 mM DTT, 10% (vol/vol) glycerol, 1 tablet of PIC per 50 ml, 4 units/ml SUPERaseIN inhibitor) on ice for 10 min and followed by one wash with the same buffer with centrifugated for 4 min at 1000x g at 4C. Cell pellets were washed twice in cell wash buffer (10 mM Tris-HCl pH 8.0, 250 mM sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 10% (vol/vol) glycerol, 1 tablet of PIC per 50 ml, 4 units/ml SUPERaseIN inhibitor). Cells were resuspended in freeze buffer (50 mM Tris-HCl, pH 8.0, 40% (vol/vol) glycerol, 5 mM MgCl2, 1.1 mM EDTA, 0.5 mM DTT and 4 units/ml RNase inhibitor) in appropriate aliquots, snap-frozen and stored at -80C until further usage. The nuclear run-on reaction was performed as a 2 biotin run-on. For this, for each sample 28 ul of the 2x Nuclear-run on master mix (10 mM Tris-Cl pH 8.0, 5 uM MgCl2, 1 mM DTT, 300 mM KCl) were mixed with 5 ul biotin-11-UTP (1 mM), 5 ul biotin-11-CTP (1 mM), 2.5 ul of each ATP and GTP (10 mM each), 5 ul H2O, 2 ul SUPERaseIN inhibitor and 50 ul of 2% Sarkosyl. The reaction mix was pre-heated at 37C and the pre-calculated number of cells added to each reaction vial, mixed thoroughly and incubated at 37C for 5 min with soft shaking (300 rpm). To stop the reaction, 350 ul of the RL Buffer from the NORGEN RNA extraction kit were added and vortexed. 240 uL 100% ethanol was added to the mixture and vortexed again. RNA extraction was performed according to the kits manual. Final RNA was eluted twice with 50 ul H2O and pooled to a final volume of 100 ul. For the base hydrolysis, the RNA was denatured for 30 s at 65C and snap-cooled on ice. 25 ul of ice-cold 1N NaOH were added and incubated on ice for 10 min. RNA was precipitated by adding 125 ul Tris-HCl (pH 6.8), 5 ul NaCl, 1 ul GlycoBlue and 650 ul 100% EtOH and centrifugation at 20,0000x g at 4C for 20 min. RNA pellet was washed with 70% EtOH, air-dried and resuspended in 6 ul H2O. For the 3' RNA adaptor ligation, 1 ul of REV3 3'RNA adaptor dilution(10 uM), heat-denatured at 65C for 20 s and placed on ice was added to the 6 ul resuspended RNA. 13 ul of the RNA ligation mix using T4 RNA ligase I was added and ..incubated at 25C for 1 hour. Biotin RNA enrichment was performed by adding 55 ul binding buffer to each ligation reaction, followed by 25 ul of pre-washed streptavidin beads. Reactions were incubated on a rotator set to 8 rpm at room temperature for 20 min. Beads were washed once with ice-cold high-salt buffer, tubes were renewed and once with low-salt buffer. On-bead 5' hydroxyl repair was prepared as follows, the beads were resuspended in 19 ul of the PNK mix and incubated at 37C for 30 min with soft shaking (350 rpm). For the 5' cap repair reaction, the beads were resuspended in the enzyme mix containing RppH and ThermoPol Reaction Buffer and incubated at 37C for 1 hour with soft shaking (350 rpm). On-bead 5' RNA adaptor ligation was prepared as follows, the beads were resuspended in 7 ul of REV5 5'RNA adaptor dilution (10 uM), heat-denatured at 65C for 20 s and placed on ice. 12 ul of the RNA ligation mix using T4 RNA ligase was added and incubated at 25C for 1 hour. Beads were washed once with ice-cold high-salt buffer, tubes were renewed and once with low-salt buffer. The RNA was cleaned-up using Trizol and chloroform. The RNA was reverse transcribed using the Maxima H minus RT enzyme and the RP1 reverse-transcription primer. The correct number of PCR cycles was determined by test PCR and Bioanalyzer analysis. In the end, the final PCR was performed using 14 cycles and the final library was cleaned-up using magnetic SPRI beads at a ratio of 1.8x and further size selected with a SPRI beads ratio of 1x to remove primer dimers.