title: GSM8633923: HeLa, without HU treatment, fixed with paraformaldehyde, input group, rep2; Homo sapiens; OTHER
library_construction_protocol: The cells were harvested by scraping, washed twice with prechilled PBS and then fixed or collected directly without fixation for further processing. When fixation was performed, the cells were subjected to crosslinking at room temperature for 10 minutes with 1% paraformaldehyde diluted in PBS, and the reaction was then quenched with 0.125 M glycine. The cells were then washed twice with prechilled DEPC-treated PBS and subsequently permeabilized with DEPC-treated PBS containing 0.25% Triton X-100 and 80 U/Ml RNase inhibitor (Biosharp, BL780B) on a rotator at room temperature for 30 minutes. After centrifugation, the cell pellets were washed once with DEPC-treated PBS. Then, fresh click reaction mixture (in DEPC-treated PBS with sequential addition and mixture of 25 μM biotin-PEG3-azide, 100 mM sodium ascorbate, 100 mM CuSO4 and 80 U/mL RNase inhibitor) was added to the pellets, which were incubated on a rotator at room temperature for 2 hours. The cell pellets were then washed once with DEPC-treated PBS and lysed on ice in lysis buffer (DEPC-treated double-distilled water (ddH2O) containing 1% SDS, 50 mM Tris-HCl (pH 8.0), and 80 U/mL RNase inhibitor) for 10 minutes. A noncontact ultrasonic disruptor (Covaris M220 focused ultrasonicator) was subsequently used to sonicate the pellets for 3 minutes to obtain smeared DNA‒RNA fragments of 100-300 bp. After sonication, the samples were centrifuged at 15000 rpm at 4°C for 10 minutes, after which the supernatant was collected. Part of the supernatant was taken as the input sample (as Total-RNA group), while the remaining portion was used as the immunoprecipitation (IP) fraction (as REFORC-RNA group). The input samples were directly extracted for RNA components and prepared for cDNA library building. The IP fraction was diluted with an equal volume of DEPC-treated PBS supplemented with cocktail and RNase inhibitor. Two hundred microliters of streptavidin beads (GE) were added to each group, and the mixtures were incubated on a rotator at 4°C for 14-16 hours. The beads were washed with DEPC-treated PBS four times. Two hundred microliters of NT2 buffer (DEPC-treated ddH2O containing 50 mM HEPES (pH 7.5), 150 mM KCl, 1 mM MgCl2, 0.05% NP-40 and 80 U of RNase inhibitor) was added to the beads in each group, which were incubated at 95°C for 5 minutes and then immediately placed on ice for 2 minutes. The beads were treated with 10 U of RQ1 RNase-Free DNase I (Promega, M6101) and 80 U of RNase inhibitor and incubated in a Thermo Mixer-Heat with rotation at 1200 rpm at 37°C for 2 hours. For the RNase H treatment group, 25 U of RNase H (NEB, M0297S) was added, and the mixture was incubated with rotation at 1200 rpm at 37°C for 1 h before DNase I treatment. Finally, 500 μL of TRIzol was added to the bead mixture, which was incubated at room temperature for 5 minutes and centrifuged at 15000 rpm for 5 minutes. Phenol–chloroform was added to the supernatant for extraction. Then, 10 μL of glycogen (20 mg/mL) and 40 μL of NaCl (5 M) were added to the RNA-containing supernatant, followed by the addition of equal volume of isopropanol. The mixture was thoroughly mixed and incubated at -80°C overnight. The next day, the mixture was centrifuged at 15000 rpm for 30 minutes at 4°C. We then washed the RNA pellet once with 75% ethanol. Finally, we resuspended the pellet in 12 μL of DEPC-treated water to prepare the sample for DNA library preparation. We used rRNA probes for rRNA cleanup. The RNA samples (no more than 2 μg) were mixed with 1 μL of rRNA probes (1 μM) and 2 μL of probe hybridization buffer (750 mM Tris-HCl, pH 8.0; 750 mM NaCl) and incubated at 95°C for 2 minutes, cooled to 22°C at a rate of 0.1°C/s and incubated at 22°C for 5 minutes. Then, 2 μL of Thermostable RNase H (NEB, M0523S), 2 μL of 10x RNase H buffer and 1 μL of ddH2O were added to the mixture, which was incubated at 50°C for 30 minutes. The RNA samples were further incubated with 2.5 μL of DNase I (NEB, M0303S), 2.5 μL of 10x DNase I buffer and 4.5 μL of ddH2O at 37°C for 30 minutes. After cleaning the rRNA, we constructed strand-specific cDNA libraries with the Hieff NGS® Ultima Dual-mode RNA Library Prep Kit (Yeasen) according to the instructions. However, we did not further fragment RNA with the reagent provided in the kit because the RNA was fragmented during sonication. The cDNA libraries were sequenced by Novogene on an Illumina platform.