Human genomic dna

Before DNA extraction, plasma samples were centrifuged at 16,000 g for 10 minutes at 4^oC in order to remove cell debris. Circulating cfDNA was extracted from 2 mL of plasma using the automated QIAsymphony extraction system and a QIAsymphony DSP Virus/Pathogen Midi kit (QIAGEN, Hilden, Germany) or using a QIAamp DNA Circulating Nucleic Acid kit (QIAGEN) according to the manufacturer’s instructions. Circulating cfDNA was eluted into 90 μL of elution buffer and stored at 4 ^oC. Eluted cfDNA was quantified by SYBR Green I real-time PCR of human LINE-1 sequences20 (link). PCR was performed in 20 μL reaction volume, containing 3 μL extracted cfDNA, 0.5 μM each of the forward primer (5′-TCACTCAAAGCCGCTCAACTAC-3′) and the reverse primer (5′-TCTGCCTTCATTTCGTTATGTACC-3′) and 1x iTaq SYBR Green Supermix (Bio-Rad, Hercules, CA). Amplification was carried out as follows: 2 minutes at 94 ^oC and 35 cycles of 10 seconds at 94 ^oC and 15 seconds at 58 ^oC and 15 seconds at 70 ^oC. A standard calibration curve was valid for 4-fold serial dilution of human genomic DNA (Promega, Madison, WI) up to 8 ng/reaction. Each sample was tested in triplicate.

All reagents and chemicals unless otherwise noted were obtained from Sigma Aldrich (St. Louis, MO, USA). All oligonucleotides and gBlocks were obtained from Integrated DNA Technologies (IDT, Corralville, IA, USA). The fluorophore-labeled oligonucleotides used for assembling the duplex OSD probe were designed to contain an inverted dT group at their 3’-ends to prevent polymerase-mediated extension (Table 1). oligonucleotides were resuspended at 100 μM concentration in TE (10:0.1, pH 7.5) buffer (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, pH 8.0) and stored at -20 °C. The concentrations of the DNA and RNA suspensions were measured by UV spectrophotometry using the NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). All enzymes including Bst 2.0 DNA polymerase and AMV RT were obtained from New England Biolabs (NEB, Ipswich, MA, USA). Human genomic DNA was obtained from Promega (Madison, WI, USA).

A PER2 promoter fragment spanning nucleotides −500 to +50 upstream of the transcription start site was synthesized from human genomic DNA (Promega) via PCR with the primers 5′-ggG cgt agt gaa tgg aag gcg-3′ and 5′-cag cag ccc aag gaa ctt-3′. The PCR products were ligated into a T&A vector (RBC Bioscience, Taipei County, Taiwan), followed by enzyme digestion with KpnI and BglII and ligation into the corresponding sites of the pGL4.2(luc2P/minP/Hygro) vector (Promega), yielding the pPER2:luc2P bioluminescence reporter. HaCaT cells were transfected with pPER2:luc2P using the Nucleofector electroporation kit (Amaxa, Inc., Köln, Germany), followed by selection with 10 μg/mL hygromycin (Sigma-Aldrich). After four weeks, a stable bioluminescence reporter cell line (HaCaT/PER2:luc2P) was obtained.

Elements were amplified via PCR from human genomic DNA (Promega) with Platinum SuperFi polymerase (Invitrogen) and primers containing attB1 and attB2 sequences (see S3 Table). Elements were then cloned into pDONR223 using Gateway BP clonase and transformed into E. coli cells. Four colonies for each element were picked and sequenced via Sanger sequencing using the RV3 primer. One clone for each element with the correct sequence was then cloned into pDEST-hSCP1-luc or pGL4-Gateway-SCP1 using Gateway LR clonase, and luciferase reporters containing the elements were then transfected into K562 cells. pGL4-Gateway-SCP1 was a gift from Alexander Stark (Addgene plasmid # 71510) [61 (link)]. To construct a positive control for the enhancer activity assays, we cloned the widely used Rous sarcoma virus promoter that has been implied to possess enhancer activities.

The methods used for the construction of the CXCL10 gene promoter-driving luciferase reporter, wild-type pCXCL10-Luc(−250/+8)) and disruption of the NF-κB-binding site by site-directed mutagenesis, pCXCR3-Luc(−178/+22)mtNF-κB, are described elsewhere [26 (link)].
For the generation of CXCR3 gene promoter-driving luciferase reporter constructs, the CXCR3 promoter fragment spanning nucleotides −178 to +22 upstream of the transcription start site was synthesized from human genomic DNA (Promega, Madison, WI, USA) via PCR using the primers 5′-ggtaccCATCCTCTGCCAGCTTTTCT-3′ (forward primer) and 5′-agatctCTTTGGTGCTTGTGGTTGGA-3′ (reverse primer). Small letters indicate inserted KpnI and BglII restriction sites. The PCR products ligated into a T&A vector (RBC Bioscience, Taipei county, Taiwan) were digested by KpnI and BglII and cloned into the KpnI and BglII sites of the pGL4-basic vector (Promega), yielding pCXCR3-Luc(−178/+22). Site-specific mutation of two NF-κB binding sites, mtNF-κB(I) and mtNF-κB(II), was performed using an EZchange Site-directed Mutagenesis Kit (Enzynomics, Daejeon, Republic of Korea), using the −178/+22 construct as a template plasmid. Primer sequences used to generate point mutations were as Table 2.
RT-PCR was carried out by annealing at 55 °C for 25 cycles. The point mutation was verified by DNA sequencing (Macrogen, Seoul, Korea).

Host RNA (18S rRNA) were quantified using the Power SYBR Green RNA-to-Ct 1-Step qRT-PCR assay (Life Technologies) and human 18S rRNA primers (5′-CCTGAGAAACGGCTACCACATC-3′ (forward), 5′-AGAGTCCTGTATTGTTATTTTTCGTCACT-3′ (reverse)). Human genomic DNA (Promega) was used as a standard control. All reactions were performed on the ABI 7900HT (Applied Biosystems).