GSM5571633: EpiLC_netcage_1; Mus musculus; OTHER
NET-CAGE library preparation was performed as described previously (Hirabayashi et al., 2019) with minor modifications. For extraction of nascent RNA, cells were firstly lysed with 1400 μl Buffer A, which is Nuclei EZ Lysis Buffer (Sigma) supplemented with 25 μM α-amanitin (Wako), 1× cOmplete Protease Inhibitor Cocktail (Roche, 4693116001) and SUPERase•IN RNase Inhibitor (20 units; Thermo Fisher Scientific), was incubated on ice for 10 min and centrifuged at 800 g for 5 min at 4°C followed by wash once with the same buffer. Washed pellets were resuspended in 200μl Buffer B, containing 1% NP-40, 20 mM HEPES pH7.5, 300 mM NaCl, 2 M urea, 0.2 mM EDTA, 1 mM dithiothreitol (DTT) (Promega), 25 μM α-amanitin, 1× cOmplete Protease Inhibitor Cocktail and SUPERase•IN RNase Inhibitor (20 units), and incubated for 10 min on ice. The suspension was centrifuged at 3000g for 2 min at 4°C. After removing the supernatant, the nuclear insoluble fraction was washed once with 100 μl Buffer B. DNase I solution (50 μl) containing DNase I (10 units; Thermo Fisher Scientific), 1× DNase I Buffer (Thermo Fisher Scientific) and SUPERase•IN RNase Inhibitor (20 units) was added to the pellets. The samples were incubated for 30 min at 37°C while being pipetted up and down several times every 10 min. QIAzol (700 μl) was then added and the solution was thoroughly mixed. RNA was extracted with an miRNeasy Mini kit (QIAGEN) according to the manufacturer's instructions. On-column DNase I digestion was carried out with an RNase-free DNase set (QIAGEN). RNA was eluted in 30 μl RNase-free water, and its quality and quantity were measured with Qubit RNA HS assay kit (Thermo Fisher Scientific) and 2100 BioAnalyzer (Agilent). cDNA was synthesized from 200 ng nascent RNA. CAGE libraries were generated according to the no amplification non-tagging CAGE libraries for Illumina next-generation sequencers (nAnT-iCAGE) protocol (Murata et al., 2014) with PCR amplifications (Takara, R060A).