title: GSM9372945: A549 WT, mock-transfected, RPB1 S2P CTD (E1Z3G) CUT&RUN-seq replicate 3; Homo sapiens; OTHER
library_construction_protocol: CUT&RUN was done according to previous reports (Miura and Chen, 2020; Morgens et al., 2025) with modifications. Briefly, 250,000 A549 WT or RNase L KO cells per well were seeded on a 12-well plate and cultured overnight before transfection. Then cells were then transfected with RNAiMAX alone or with 0.76 μg/ml poly(I:C) for 4 hours The cells were permeabilize on plate with 500μL Perm buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine (Sigma, S2626), 0.1% Triton X-100 (Sigma, X100), and proteinase inhibitor (Roche, 04693132001)) for 15 minutes at room temperature. The cells were washed again with Perm buffer then incubated with either anti-Rpb1 S5P CTD (4H8, Cell Signaling, cat #2629) at 1: 50 dilution, equal mass of mouse normal IgG (Santa Cruz, cat # sc-2025), or anti-Rpb1 S2P CTD (clone E1Z3G, Cell Signaling, cat # 13499) at 1: 50 dilution, in 200μL Perm buffer for 1 hour at room temperature. The cells were then washed twice with Perm buffer and incubated with pAG-MNase (Cell Signaling, cat #40366) at 1:33 dilution in 200μL Perm buffer for 1 hour at room temperature.  The cells were then washed twice with Perm buffer and chill on ice. Then 225 μL ice-cold Perm buffer containing 5 mM CaCl2 was dispensed per well, and the plate was incubated in a refrigerator at 4°C for 30 min for targeted digestion. The reaction was halted by adding 75 μL 4× STOP solution (680 mM NaCl, 40 mM EDTA, 8 mM EGTA, 100 μg/ml RNase A (Invitrogen, cat # 12091021) and 0.1% Triton X-100, spiked with yeast genomic DNA (Cell Signaling, cat #40366) at 10pg per sample for 4H8 and IgG, 30pg per sample for E1Z3G). The plate was then incubated at 37°C for 30min to facilitate fragment release. The supernatant was collected and then 3μL 20% SDS solution and 3.75μL 20mg/ml proteinase K solution was added. The mixture was then incubated at 50°C for 1 hour. The DNA was extracted with phenol:chloroform. The sequencing library was constructed with NEBNext® Ultra™ II DNA Library Prep Kit (NEB, cat # E7645L) following a previous report (Liu et al., 2018a) with modifications. Briefly, equal volume of purified DNA fragments was used. End prep was performed at 20°C for 30 min and then 50°C for 60min to prevent short fragment melting. Fragments were then ligated to adaptors from NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 1, NEB, cat # E7395S) and cleaned up twice with 1.1X Ampure XP beads (Beckman Coutler, cat # A63880). The resulting ligated products were then PCR amplified for 10 cycles for 4H8 and E1Z3G, and 14 cycles for IgG samples, respectively. The cycle numbers were determined empirically by qPCR. The amplified libraries were double-sided size selected with 0.6X and 1.1X Ampure XP beads and further cleaned up by 1 round with 1.1X bead for 4H8, E1Z3G, or 3 rounds for IgG samples, to remove adaptor dimers.  The libraries were sequenced on AVITI platform (Element Biosciences) by UC Davis sequencing core.