GSM5381364: HBECs_2 (Nanopore); Homo sapiens; RNA-Seq
After media removal, cells were trypsinized and washed with PBS. We preformed nuclei fractionation before nascent RNA purification. Briefly, cells were lysed for 2 min with 400 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), layered over 1000 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. All steps were performed at 4 °C and all buffers were prepared with 0.05U/μl SUPERase.In (ThermoFisher Scientific, AM2694) and protease inhibitor mix (Roche, 11873580001). extract the RNA from nucleus with Trizol. 1ml douncer was used to homogenize the nuclei. 4sU labeled nascent RNA was purified from the nucleus RNA (methods described in session: 4sU metabolic labeling and nascent RNA sequencing). polyA tailing was done with 5 unit Ecoli polyA polymerase in 20ml volume, incubated at 37°C for 30min. 0.5 ml RNase inhibitor was added to prevent RNA degradation. Two RNA sequencing libraries were prepared from 500 ng polyadenylated nascent RNA using the Nanopore Direct RNA Sequencing kit (SQK-RNA002, Oxford Nanopore Technologies) according to manufacturer's instructions. Briefly, the poly(T) adapter was ligated to the RNA using T4 DNA ligase (New England Biolabs, M0202) in the Quick Ligase reaction buffer (New England Biolabs, B6058) for 10 min at room temperature. SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, 18080044) was used to synthesize the first-strand cDNA. The RNA–cDNA hybrid was then purified using 1.8X Agencourt RNAClean XP magnetic beads (Beckman Coulter, A66514). Finally, the RNA sequencing adapter was ligated onto the RNA-cDNA hybrid using T4 DNA ligase in the Quick Ligase reaction buffer for 10 min at room temperature, and the final library was purified using 1X Agencourt RNAClean XP magnetic beads. Each library was loaded onto a flow cell (FLO-MIN106, R9.4.1) and sequenced using a MinION sequencer (Oxford Nanopore Technologies) during 20 – 23 hour long runs. The direct RNA sequencing raw data was processed using Guppy version 4.0. The sequencing reads were mapped to human reference genome hg19 using minimap2 version 2.17. The aligned read bam files were indexed and visually inspected using IGV.