title: GSM8660254: INTS4K/A, PRO-seq, Rep1; Homo sapiens; OTHER
library_construction_protocol: For permeabilization, a 80% confluent plate of HBEC was washed with PBS to remove media before lightly trypsinization and transfer to cold PBS. Cells were maintained at 4°C for the rest of the permeabilization steps. Cells were spun at 200 g for 3 min and suspended in 10 ml PBS for counting. Cells were re-spun at 200 g for 3 min and suspended in 250l of Buffer W (10 mM Tris-Cl, pH 8.0, 10 mM KCl, 250 mM Sucrose, 5 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 10 % (v/v) Glycerol, 0.5mM DTT,  0.2l /mL SUPERase-in RNase inhibitor) before 10 ml of Buffer P (Buffer W + 0.05 % (v/v) Tween-20, 10 % (v/v)) was slowly added from the side of the tube and incubated for 5 min. Thereafter cells were spun down at 300 g for 3 min for wash with 10ml of Buffer W and re-spun for final suspension in 400 of Buffer F (50 mM Tris-Cl, pH 8.0, 40 % (v/v) glycerol, 5 mM MgCl2, 1.1 mM EDTA, 0.5 mM DTT, DTT to 0.5mM + SUPERase-IN RNase inhibitor to 1 L/mL final) before final counting.  For run-on, the volume of cell suspension was adjusted to 1 million cells in 45 l of Buffer F and 50,000 drosophila S2 cells in 5 l of Buffer F were added as spike-in. 25 L of sarkosyl was mixed with preheated 25 L of 4x run-on mix ( 20 mM Tris-Cl pH 8, 20 mM MgCl2, 2 mM DTT, 600 mM KCl, 40 M of biotin-11-NTP and 11 L nuclear free water) at 37°C. The permeabilized cells were incubated at 37°C before incubating with the complete run-on mix for 5 min at 37°C. Run-on was stopped by vortexing with Buffer RL (Norgen Biotek # 37500) for 10 min. 240 L of ethanol was further added for RNA purification with Norgen column (Norgen Biotek # 37500) using the manufacturer's protocol. Briefly, the run-on lysate was passed through the column and eluted in 50 L of water after washing thrice with solution A (Norgen Biotek # 37500). The sample was incubated with 25 L of ice-cold fragmentation buffer (375 mM Tris·Cl, pH 8.3, 562.5 mM KCl, 22.5 mM MgCl2) at 95°C for 5 min and reaction stopped with 125 L of 0.1 M EDTA before incubating on ice.  For adding 3' adapter, RNA binding beads were prepared as follows: 20 l MyOne Streptavidin C1 DynaBeads (Thermo # 65001) per samples were prepared by resuspending and rotating 2 min for three times in Decon buffer (0.1 M NaOH, 100 mM NaCl) before washing twice in succession with 100 mM NaCl and Binding buffer (10 mM 1 M Tris-Cl, pH 7.4 , 300 mM NaCl, 0.1% Triton X-100, 1 mM EDTA and 0.2 µl SUPERase⋅In per ml) and finally suspended in 100 l of Binding buffer. 50 µl these beads were added to the sample and washed as follows: 2× high-salt buffer (low-salt + 2M NaCl), 2× binding buffer and 2× low-salt buffer (5 mM Tris-Cl, pH 7.4, 0.1% Triton X-100, 1 mM EDTA) before resuspending in 500 µl TRIzol. The sample was incubated at 65°C for 5 min with intermitted vortexing. The RNA was extracted from the aqueous layer by chloroform extraction and ethanol precipitation. The pellet was washed with 75% ethanol and air dried. This pellet was suspended in 5 l of 2.5mM 3' adapter (App-VRA3-6N) and incubated for 30 s at 65°C. 15 l of ligation master mix (6 µl 50% PEG 8000, 2 µl 10× T4 RNA ligase buffer, 0.5 µl SUPERase⋅In, 1 µl T4 RNA ligase 2, truncated KQ, and 5.5 µl water) was added and incubated overnight at 16°C.  For 5' end processing, 180 ml of betaine buffer (1.25 M in Binding buffer) was added to the ligated mix and incubated for 5 min at 65°C before adding the remaining beads for rotation at room temperature for 20 min. Beads were washed as follows: 2× binding buffer, 2× high-salt buffer and 2× low-salt buffer. Decaping was done by incubating in MDE master mix for 30 min at 37°C after washing once with MDE buffer (New England Biolabs #M0608S). Subsequent 5' hydroxyl repair was done by washing the beads as follows: 2× high-salt buffer, 2× low-salt buffer and 2× PNK buffer (New England Biolabs M0201) before incubating with PNK master mix (13 µl water, 2 µl 10× PNK buffer, 2 µl 10 mM ATP, 1 µl of T4 PNK and 1 µl SUPERase) for 1 h at 37°C. For 5' adapter ligation, beads were washed as follows: 2× high-salt buffer, 2× binding buffer, 2× low-salt buffer and 1× with 0.25X SSIV buffer (Invitrogen, cat. no. 18090200) before incubating with 7.5 µl RT mix (3 µl 5× SSIV buffer, 0.75 µl 0.1 M DTT, 0.75 µl, 12.5 mM dNTP mix, 0.75 µl SUPERase⋅In, 1 µl SSIV RTase, 1.25 µl water) in a thermocycler following the temperature program: 10 min at 48°C, 15 min at 54°C, hold at 4°C). PRO-seq libraries were generated using Ultra II Q5 Master Mix (New England Biolabs # M0544) following the manufacturer's protocol.