GSM3318256: NET-CAGE_mouse_brain_biologicalRep1_technicalRep1; Mus musculus; 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.). Frozen mouse tissue samples were used for total and nascent RNA extraction. Tissue was homogenized using gentleMACS Dissociator (Miltenyi Biotec).  The sample was transferred to gentleMACS M tube (Miltenyi Biotec) and added with pre-chilled 2 ml of Buffer C (Nuclei PURE Lysis Buffer (Sigma-Aldrich), 25 μM alpha-amanitin, 1× cOmplete Protease Inhibitor solution, 20 units of SUPERase IN RNase Inhibitor, 1 mM DTT, Triton X-100). Lysate was then filtered through gentleMACS SmartStrainers (100 μm) (Miltenyi Biotec). 200 μL of the tissue lysate was sampled and mixed with 700 μL of QIAzol for total RNA extraction. We added 2 ml of ice-cold Buffer D (10 ml of Sucrose Cushion solution (Sigma-Aldrich), 1.2 ml of Sucrose Cushion buffer (Sigma-Aldrich) and 11.2 μL of 1M DTT) to the bottom of a conical centrifuge tube on ice. Tissue lysate prepared above was gently mixed with 3.6 ml of Buffer D, and was carefully and slowly layered on top of the 2 ml of Buffer D in the conical centrifuge tube. Then, the tube was centrifuged at 13,000 G for 45 minutes at 4 ˚C to collect nuclei pellets. The pellets were washed once with 500 μL of Buffer C. 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).