GSM3318218: NET-CAGE_GM12878_8M_biologicalRep1; Homo sapiens; OTHER
The cDNA synthesis was carried out using 5 μg of total and nascent RNA (A260/280 and 260/230 ratios > 1.7). CAGE libraries were generated based on no-amplification non-tagging CAGE libraries for Illumina next-generation sequencers (nAnT-iCAGE) precisely as described in Murata et al. (Methods Mol Biol. 2014;1164:67-85.). Approximately 1.0 × 107 cells were used for total and nascent RNA fractionation. Culture medium was transferred to a conical centrifuge tube and centrifuged at 800 G for 5 minutes at 4 ˚C. The supernatant was removed and the cell pellets were washed with 5 mL of ice-cold D-PBS. To lyse cells, 1,000 μL of Buffer A (Nuclei ez lysis buffer (Sigma-Aldrich), 25 μM alpha-amanitin (FUJIFILM Wako Pure Chemical), 1× cOmplete Protease Inhibitor solution (Sigma-Aldrich) and 20 units of SUPERase IN RNase Inhibitor (Thermo Fisher Scientific)) was added to the cell pellets and resuspended by gentle vortex. 100 μL of the cell lysate was sampled and mixed with 700 μL of QIAzol for total RNA extraction. The remaining lysate was set on ice for 10 minutes. Then the lysate was centrifuged at 800 G for 5 minutes at 4 ˚C. The pellets were washed once with 600 μL of Buffer A. Washed pellets were resuspended in 200 μL of Buffer B (1% NP-40, 20 mM HEPES, 300 mM NaCl, 2 M Urea, 0.2 mM EDTA, 1 mM DTT, 25 μM alpha-amanitin, 1× cOmplete Protease Inhibitor solution and 20 units SUPERase IN RNase Inhibitor), and incubated for 10 minutes on ice. Then, nuclear insoluble fraction was collected by 3,000 G centrifuge for 2 minutes at 4 ˚C. The pellets were washed once with 100 μL of Buffer B. 50 μL of DNase I solution containing 10 units of DNase I (Thermo Fisher Scientific), 1× DNase I Buffer (Thermo Fisher Scientific) and 20 units of SUPERase IN RNase Inhibitor was added to the pellets. The solution was incubated for 30 minutes at 37 ˚C while pipetting up and down several times every 10 minutes. 700 μL of QIAzol was directly added to the solution and thoroughly mixed. RNA was extracted by miRNeasy Mini kit (QIAGEN) according to the manufacturer's instructions. An “on-column DNase I digestion” was carried out using RNase-free DNase set (QIAGEN). RNA was eluted in 30 μL of RNase-free water, and quality and quantity were measured by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and 2100 BioAnalyzer (Agilent).