title: GSM3731637: S2_ONT_DMSO_2; Drosophila melanogaster; OTHER
library_construction_protocol: S2 cells in an adherent layer at 95% confluency were lifted with gentle pipetting and proceeded imediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 50 million S2 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed 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) before pulse vortex and incubation 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 all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. 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 (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylacohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254). Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using RiboMinus Eukaryotic Kit v2 (ThermoFisher, A15020). A poly(A) tail was added to the 3' ends of the RNA sample with E. coli poly(A) polymerase (Clontech/Takara, 2180), incubating for 7.5 min at 37°C. Direct RNA sequencing protocol using the SQK-RNA002 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software for 48 hours with default settings for direct RNA sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3731637