GSM1438941
GSM1438941: GRO-seq_TNFα_40m_rep2; Homo sapiens; RNA-Seq
MCF-7 cells were seeded at 2.5 million cells per 15 cm dish and treated with Vehicle, E2, TNFα, or E2 + TNFα, as described above.  The cells were washed three times with ice-cold PBS, swollen in hypotonic buffer, and collected in ice-cold lysis buffer [10 mM Tris•HCl, pH 7.4, 0.5% NP-40, 10% Glycerol, 3 mM CaCl2, 2 mM MgCl2, 1 mM DTT, 1X protease inhibitor cocktail (Sigma-Aldrich), and SUPERase-In (Ambion)] at 1000 x g for 10 min at 4°C.  The cells were then resuspended in 1.5 ml of lysis buffer and pipetted up and down through a narrow opening 30 to 50 times to release the nuclei.  The nuclei were pelleted again by centrifugation and washed once with 1 mL of lysis buffer.  The nuclear pellets were resuspended in 500 μL of freezing buffer (50 mM Tris•HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, and 4 units of SUPERase-In per mL), counted, and stored in 100 μl aliquots containing 5 x 106 nuclei. Nuclear run-on and library preparation was performed as previously described (Danko et al., 2013; Hah et al., 2011), with modifications.  Briefly, cells were treated for 40 min with 100 nM E2, 25 ng/ml TNFα, or both E2 + TNFα and libraries were prepared from two biological replicates using a circularized ligation-based protocol for adaptor addition used to improve the efficiency of library preparation, reduce sequence bias, and allow for barcoding.  The libraries were amplified with indexed primers containing barcodes according to the Illumina TrueSeq small-RNA library prep kit, then sequenced using an Illumina HiSeq 2000.  A more detailed protocol is available from the corresponding author (W.L.K.).
GEO
GSM1438941
GEO Accession
GSM1438941