GSM8759277: HCT116-siCtrl-KAS-seq-Input-rp1; Homo sapiens; OTHER
RNA was isolated using Trizol method and precipitated using ethanol. BID-seq for nucleus RNA: Nucleus fraction was isolated based on cell fractionation protocol. Nucleus RNA was purified by TRIzol reagent (Invitrogen) and ethanol precipitation. rRNA was further removed from Nucleus RNA by RiboMinus™ Eukaryote System v2 (Invitrogen). 50 ng nucleus RNA from PUS7KD and control HCT116 cells were used for BID-seq. The BID-seq was performed as previously described. 3 biological replicates were used for both the PUS7 KD and control conditions. PAR-CLIP: We performed PAR-CLIP based on a previously reported protocol. Briefly, four 15 cm plates of HCT116 cells were seeded per replicate and grown to 80% confluency before adding 4 μL of 1M 4SU to each plate. After 14 hours of incubation, the media was aspirated, and the cells were washed once with 5 mL of ice-cold PBS per plate. The cells were crosslinked on ice using 0.15 J cm⁻² of 365 nm UV light, applied twice. Crosslinked cells were collected with cell lifters, and four volumes of lysis buffer (50 mM HEPES, pH 7.5; 150 mM KCl; 2 mM EDTA; 0.5% [v/v] NP-40; freshly added 1:100 protease inhibitor (Roche)) were added to the pellet. After 10 minutes of incubation on ice with periodic mixing, the lysate was centrifuged at 15,000 g for 15 minutes, and the supernatant was collected. RNase T1 (Thermo Scientific) was added to a final concentration of 0.1 U μL⁻¹, followed by a 15-minute incubation at room temperature, which was then quenched on ice. 5 µg of anti-PUS7 were added to the lysate and incubated at 4 °C for 2 hours with periodic rotation. 100 µL Protein A or G beads (Thermo Scientific) were washed twice with IP wash buffer (50 mM HEPES, pH 7.5; 300 mM KCl; 0.05% [v/v] NP-40; freshly added 1:100 protease inhibitor (Roche) and 40 U mL⁻¹ RNasin®) and resuspended in 50 μL lysis buffer before being added to the antibody-lysate mixture. Another 2-hour incubation at 4 °C with low-speed rotation followed. After incubation, the beads were washed three times with IP wash buffer, resuspended in 200 μL of IP wash buffer, and subjected to a second RNase T1 digestion (10 U μL⁻¹ final concentration) for 15 minutes at room temperature. The reaction was quenched with 10 μL SUPERase-In, followed by a 5-minute incubation on ice. Beads were washed three times with high-salt wash buffer (50 mM HEPES, pH 7.5; 500 mM KCl; 0.05% [v/v] NP-40; freshly added 1:100 protease inhibitor (Roche)) and twice with 1X PNK buffer (NEB). They were resuspended in 200 μL of 1X PNK buffer and underwent T4 PNK end repair (Thermo Fisher Scientific) at 37 °C using standard procedures. After incubation, beads were washed once with 1X PNK buffer, followed by proteinase K digestion. RNA was recovered using RNA Clean & Concentrator (Zymo Research) and prepared for library construction using the NEBNext Small RNA Library Prep Set for Illumina (NEB). Alternative adaptors were used for small RNA library preparation. 3′ SR Adapter: 5′App-NNNNNATCACGAGATCGGAAGAGCACACGTCT-3SpC3; 5′ SR Adapter: 5′-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3′. The libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina Nova-seq 6000 platform. KAS-seq: KAS-seq was performed following a previously reported protocol. Briefly, HCT116 cells were transfected with siPUS7, 7SK ASO, or siCtrl and harvested into 1.5 mL tubes after 3 days. Cells were suspended in 1 mL medium containing 500 mM N3-kethoxal and incubated on a shaker at 37 °C for 10 minutes to label genomic ssDNA. The medium was then discarded, and genomic DNA was extracted. The N3-kethoxal-labeled ssDNA was biotinylated, enriched, and fragmented according to the established KAS-seq protocol. Dual-index libraries were prepared for high-throughput sequencing using the Accel-NGS Methyl-seq DNA Library Kit and sequenced on Illumina Nova-seq 6000 platform. mRNA-seq: HCT116 cells were transfected with siPUS7, 7SK ASO, or siCtrl and harvested into 1.5 mL tubes after 3 days. mRNA was extracted and applied to mRNA-seq using NEBNext® Ultra™ II RNA Library Prep Kit (NEB). The libraries were sequenced on Illumina Nova-seq 6000 platform.