GSM8791274: K562_timecourse_untx_SLAMRTTag_IgG_n5_(230111,231128_NK_hs_1661); Homo sapiens; OTHER
K562 cells were cultured until they reached approximately 70% confluency. For RNA metabolic labeling, 100 µM s4U was added to the cell media for 4 hours. Cells were then collected by centrifugation at 300 g for 5 minutes and washed with 1x PBS. Nuclei were isolated by resuspending the cell pellets in NE1 buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 0.5 mM spermidine, Roche Complete Protease Inhibitor Cocktail, 1 U/μL RNasin Ribonuclease Inhibitor) and incubating on ice for 10 minutes. The nuclei were centrifuged at 500 g for 8 minutes and resuspended in Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, Roche Complete Protease Inhibitor Cocktail, 1 U/μL RNasin Ribonuclease Inhibitor).  Next, 200,000 K562 nuclei were bound to 5 μL of ConA beads (Bangs Laboratories) for 10 minutes at room temperature. After removing the Wash Buffer, the nuclei were treated with 10 mM iodoacetamide in Wash Buffer and incubated for 1 hour at 37°C. The nuclei were then washed twice with Wash Buffer.  Subsequently, standard RT&Tag steps were followed starting from primary antibody incubation overnight. The detailed RT&Tag protocol can be found here: dx.doi.org/10.17504/protocols.io.x54v9jyjqg3e/v1. Sequencing libraries were then amplified by mixing the nuclei suspension with 15 μL of 0.67% Triton X-100, 2 μL of 10 mM i7 primer, 2 μL of 10 mM i5 primer, and 25 μL of 2x NEBNext Master Mix (NEB). The following PCR conditions were used: 1) 58°C for 5 minutes, 2) 72°C for 5 minutes, 3) 98°C for 30 seconds, 4) 98°C for 10 seconds, 5) 60°C for 15 seconds, 6) Repeat steps 4-5 13 times, 7) 72°C for 2 minutes, 8) Hold at 4°C. The sequencing libraries were then purified using 0.8x HighPrep PCR Cleanup System (MagBio) beads according to the manufacturer's instructions. Sequencing was performed using single-end 100 bp reads.