GSM3318201: NET-CAGE_MCF-7_2M_frozen_DMSO_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 x 107 cells were prepared for different freezing conditions as follows. (i) Fresh: fresh MCF-7 cell pellets without cryopreservation were used as a control. 1,000 μL of Buffer A was directly added to the fresh cell pellets. (ii) Flash-frozen (Cryopreservation in pellet form): cell pellets were flash-frozen. Pellets without any medium were stored at -80 ˚C for 4 days. 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 directly added to the frozen cell pellets. (iii) Cryopreservation with 10% Dimethyl sulfoxide (DMSO): the pellets were resuspended in 5 ml of DMEM containing 10% DMSO supplemented with FBS. The suspension was slowly frozen and stored for 4 days at -80 ˚C. Frozen cells were quickly thawed at 37 ˚C in a water bath. Then the suspension was centrifuged at 800 G for 5 minutes at 4 ˚C and the supernatant was removed. 1,000 μL of Buffer A was added to the cell pellets. (iv) Cryopreservation with CELLBANKER® 1 plus (NIPPON ZENYAKU KOGYO): the pellets were resuspended in 5 ml of CELLBANKER® 1 plus. The suspension was frozen at -80 ˚C and stored at -80 ˚C for 4 days. Frozen cells were quickly thawed at 37 ˚C in a water bath. Then the suspension was centrifuged at 800 G for 5 minutes at 4 ˚C and the supernatant was removed. 1,000 μL of Buffer A was added to the cell pellets. The 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).