Illumina HiSeq 2500 paired end sequencing; Studying enhancer activity by FAIRE-STARR-seq in the U2OS-GR and the U2OS-AR cells upon hormone treatment
Studying enhancer activity by FAIRE-STARR-seq in the U2OS-GR and the U2OS-AR cells upon hormone treatment
RNA was extracted from treated cells using the RNeasy midi kit from Qiagen. GR18 cells were grown to approximately 90% confluence according to standard protocols in DMEM medium with 5% FBS 5 million cells were transfected with 5 µg of DNA FAIRE-STARR library plasmid using the Amaxa Nucleofector kit V (Lonza). For each condition, 4 transfections were pooled and seeded into two 15cm dishes with medium containing PKR (C16, Sigma; cat# I9785-5MG) and TBK1/IKK inhibitors (BX-795, Sigma; cat# SML0694-5MG) at a final concentration of 0.5 µM per inhibitor to suppress the type I interferon response (PMID 29256496). U2OS-GR cells were treated with 1 µM Dex or 0.1 % EtOH as a vehicle control. U2OS-AR cells were treated with 5 nM R1881 or 0.1 % DMSO as vehicle control. The poly(A) fraction was isolated from total RNA using oligo d(T) beads. RNA was treated with TurboDNase (Thermo Fisher Scientific) for 30 minutes at 37°C and cleaned up using the RNA Agencourt RNAclean XP (Beckman Coulter). A modified primer (5'- CAAGCAGAAGACGGCATACGAGATnnnnnnnnGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT -3') was used during the reverse transcription step to introduce unique molecular identifiers (UMIs). 50 ng cDNA/reaction was used as a template in every 50 μL PCR reaction using  Kapa Hifi hotstart ready mix (Roche). PCR conditions: 98 °C for 45 s; 15 cycles of 98 °C for 15 s, 65 °C for 30 s and 72 °C for 30 s. Illumina HiSeq-compatible primers were used (forward: 5'- CAAGCAGAAGACGGCATACGA-3'; reverse: 5'- AATGATACGGCGACCACCGAGATCTACAC-index-ACACTCTTTCCCTACACGACGCTC-3'). PCR products were purified with AMPure XP beads (Beckman Coulter; beads/reaction ratio = 1)