title: GSM2151690: RNA-seq_PARP-1KD_MCF7_2; Homo sapiens; RNA-Seq
library_construction_protocol: GRO-seq: MCF-7 cells were washed three times with ice-cold PBS and then resuspended for swelling in ice-cold Hypotonic Lysis Buffer [10 mM Tris•HCl, pH 7.4, 0.5% NP-40, 10% Glycerol, 3 mM CaCl2, 2 mM MgCl2, and 1 mM DTT containing 1x protease inhibitor cocktail (Sigma-Aldrich) and 4 units/mL SUPERase-In (Ambion)].   The swollen cells were collected by centrifugation at 1000 RCF for 10 min at 4°C and then resuspended in 1.5 ml of lysis buffer and pipetted up and down through a narrow opening tip 30 to 50 times to lyse the cells and release the nuclei.  The nuclei were collected by centrifugation and washed once with 1 mL of Hypotonic Lysis Buffer.  After a final collection by centrifugation, the resulting pellets of nuclei 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/mL of SUPERase-In per mL), counted, frozen in liquid nitrogen in 100 μL aliquots containing 5 x 106 nuclei, and stored at -80°C until use. RNA-seq: Following 3 hours of treatment, RNA was harvested using QIAGEN RNeasy Kit. GRO-seq: Nuclear run-on and GRO-seq library preparation were performed as previously described {Core, 2008;Hah, 2011}.  Briefly, nuclear run-on reactions were performed for ~100 bases in the presence of sarksoyl (to prevent reengagement of RNA polymerases), rNTPs, α32P-CTP, and 5-bromo-UTP.  The nascent RNAs were isolated, hydrolyzed to ~100 bases, and enriched using α-BrdUTP antibody-conjugated agarose beads (Santa Cruz).  The bound RNAs were washed several times and eluted.  The 5’ RNA cap was removed and the ends were repaired in preparation for adapter ligation.  Small RNA adapters were ligated to the 5’ end, followed by another bead binding enrichment using α-BrdUTP antibody-conjugated agarose beads.  These steps were repeated using a 3’ adapter.  The resulting RNAs were reverse transcribed, amplified using PCR, and analyzed by high throughput sequencing using an Illumina 1G Genome Analyzer. RNA-seq: RNA was prepared for sequencing as previously described (Sun, M. et al. Mol. Cell 2015).
attributes.tag: GEO Accession
attributes.value: GSM2151690