NextSeq 500 sequencing; Requirement for the Mediator kinase module for activation of super-enhancer associated genes in colon cancer cells
Requirement for the Mediator kinase module for activation of super-enhancer associated genes in colon cancer cells
Total RNA was collected 3 days after the initial siRNA transfection. Human colon cancer HCT116 cells were cultured in McCoys 5A media (Gibco) supplemented with 10% FBS (Gibco), L-glutamine and antibiotics.  Cells were seeded on 6-well plates, 3 replicates/transfection, for experiments 1 day before transfection. Cells were transfected with Dharmacon SMARTpool ON-TARGETplus siRNA (20 pmol/target/1 ml of media CDK8: L-003242-00, CDK19: L-004689-00, MED12: L-009092-00, MED13: L-019908-00, MED13L: L-027126-00, BRD4: L-004937-00, NT: D-001810-10) in antibiotic-free medium using Lipofectamine RNAi Max according to standard protocols and media was changed 4 h later. siNTpool was used to achieve the same final amount of siRNAs in single knockdowns compared to combination knockdowns. The transfection was repeated the following day. RNA was extracted using Qiagen RNeasy Plus standard methods with additional DNase treatment (Qiagen RNase-free DNase set). Total RNA (2,5μg) was treated with Illumina Ribo-Zero rRNA Removal reagent to remove ribosomal RNA according to Ribo-Zero Magnetic Kits Guide Part # 15065382 Rev A November 2014protocol. cDNA libraries were generated from 10 ng ribosomal RNA depleted RNA using NEBNext Ultra Directional RNA Library Prep Kit according to the following protocols: NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina® Instruction Manual (Protocol to use with Purified mRNA or Ribosome Depleted RNA) #E7420S/L, NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) Instruction Manual #E7335S/L, NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 2) Instruction Manual #7500S/L,  Denaturing and Diluting Libraries for the NextSeq 500 Part# 15048776 Rev. D October 2014 : loading concentration 1,3 pM, + 1% PhiX control
Experimental Factor: RNA interference
siCDK8 + siNT