title: GSM8059166: HeLa 4sU labelled chromatin-associated RNA - minA; Homo sapiens; RNA-Seq
library_construction_protocol: Plates were washed twice with 1X PBS and cells were lifted by scraping. Labeled cells were collected by centrifugation at 500 g for 2 minutes. Nuclei were collected by lysing samples of 10M cells in 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40 (Thermo Fisher Scientific, 28324), 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl) for 2 min, layering over a 500 μl sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and centrifuging at 16,000 g for 10 minutes. The nuclei pellet was washed in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifuging at 1,150 g for 1 minute. Nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) by pulse vortex and incubated on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and buffers were prepared with 25 μM α-amanitin (Sigma, A2263), 0.05U/μl SUPERase.In (ThermoFisher Scientific, AM2694), and protease inhibitor mix (Roche, 11873580001). Chromatin-associated RNA was extracted using Qiazol lysis reagent (Qiagen, 79306) following the manufacturer's instructions. For more details about cell harvest, please see (Smith et al 1998).  50ug RNA were subjected to 4sU purification. In brief, labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1 mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours 24°C in the dark and 800 rpm to mix. RNA was purified by shaking the sample with a 1:1 volume of chloroform/isoamylacohol (24:1), separating using a phase-lock tube at 16,000 g for 5 min, and performing isopropanol precipitation. For more detials, please see (Girard et al 2011) and (Wang et al 1990). Biotinylated RNA was separated using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101) by mixing with μMACS streptavidin beads at a 2:1 ratio by volume at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix was transferred to the μMACS column and washed with wash buffer (100 mM Tris pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20) at 65°C and room temperature 3 times each. Selected RNA was eluted off the magnet using the reducing agent, DTT (0.1M), and purified using the miRNeasy micro kit (Qiagen, 217084) with on-column DNase I treatment (Qiagen, 79254). For the poly(A) depleted sample, the RNA was first concentrated using the RNA Clean and Concentrator kit (ZymoResearch, R1015). 10 μL Oligo(dT) Dynabeads (ThermoFisher, 61002) were washed in 10 μL Binding Buffer (20mM Tris.HCl pH 7.5, 1M LiCl, and 2mM EDTA). The sample was mixed with 10 μl binding buffer, heated to 65°C for 2 min, moved to ice, and mixed with 1 μl SUPERase.In (ThermoFisher Scientific, AM2694). The sample and beads were mixed thoroughly and annealed by rotating continuously on a mixer for 5 minutes at room temperature. Poly(A) RNAs were collected on a magnet while depleted supernatant was removed and purified using the RNA Clean and Concentrator kit (ZymoResearch, R1015). Illumina sequencing libraries were prepared using the Ovation Universal RNA-seq System (NUGEN, 0343-32) with Universal Human rRNA strand selection reagent (NUGEN, S01859) following the manufacturer's instructions.