For CLASH, the cells were washed off the filters using 25mL of cold phosphate buffer saline and pelleted by centrifugation. Cells were subsequently resuspended in 2 volumes of TN150-lysostaphin buffer (50 mM Tris pH 7.8, 150 mM NaCl, 100 µg/mL lysostaphin, 0.1% NP-40, 0.5% Triton X-100) and transferred to 5 mL screw-cap tubes (Eppendorf). Sixty µL of DNase RQ1 and 10 µL of SUPERase-In was added and the mixture was incubated at 37°C for 30 min to lyse the cells. Afterwards, 3 volumes/cell weight of zirconia beads (Thistle Scientific; 0.1 µm) were added and the mixture was vortexed vigorously 5 times for one minute with incubation on ice between each samples. Two volumes/cell weight of cold TN150 anti-peptidase (50 mM Tris pH 7.8, 150 mM NaCl, 1 Roche EDTA-free mini pellet, 0.1% NP-40, 0.5% Triton X-100 and 10 mM EDTA) was added before centrifugation for 30 minutes at 10000 g at 4°C. Subsequently, 75 µL of anti-FLAG® M2 Magnetic Beads (pre-washed with TN150 buffer) was added and incubated with the lysate for 2 hours at 4°C. The beads were then washed three times for five minutes with 2 mL of TN1000 buffer (50 mM Tris pH 7.8, 1M NaCl, 0.1% NP-40, 0.5% Triton X-100) and three times with 2 mL of TN150 buffer (50 mM Tris pH 7.8, 150 mM NaCl, 0.1% NP-40, 0.5% Triton X-100) for 5 minutes. Beads were subsequently resuspended in 250 µL of TN150 and 10 µL of homemade GST-TEV protease was added. The samples were rotated for 2 hours at room temperature. TN150 was added to a final volume of 600 µL and 550 µL of the TEV eluates were incubated with RNace-it™ (Agilent Technologies, 1 µL of 1:100 dilution) for exactly seven minutes at 20°C. Subsequently, 500 µL of the mixture was added to a tube with 0.4 g guanidium-HCl and vortexed vigorously to inactivate the RNases. Sodium chloride and imidazole (pH 8.0) were added to a final concentration of 300 mM and 10 mM respectively, and the mixture was transferred to 50 µL of Ni-NTA agarose beads (QIAGEN), pre-equilibrated with wash buffer I (300 mM NaCl, 10 mM imidazole, 6M GuHCl, 50 mM Tris-HCl pH 7.8, 0.1% NP40, 5 mM β- mercaptoethanol and 0.5% Triton X-100) and incubated at 4°C overnight on a rotator. The next day the beads were transferred to Pierce snap-cap columns (Thermo Scientific) and washed twice with 500 µL wash buffer I and three times with 500 µL 1x NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% NP40, 5 mM βMe and 0.5% Triton X-100). For RNAtag-Seq, total RNA was extracted from the cells using standard guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski et al, 1987). For CLASH, the isolated RNA-RNA complexes had 3' and 5' Illumina adaptors ligated on their respective ends as described previously (McKellar et al, 2020; Granneman et al, 2009). For RNAtag-Seq, the total RNA samples were processed as described previously (Shishkin et al, 2015).