# Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3
## 4 Materials and Methods
### Generating IRAK3 Gene Knockdown THP-1 Cells  
Two knockdown cell lines were developed using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 cleavage system. Based on human IRAK3 sequence (NG_021194.1) from the NCBI database, single guide RNA (sgRNA) sequences were designed to target IRAK3 gene using the Genetic Perturbation Platform (GFP) sgRNA Design Software (Broad Institute), known as sgRNA1 and sgRNA2 (Supplemental Table S2). An equimolar ratio of sgRNA1, sgRNA2 (Thermo Fisher Scientific) and Cas9-GFP protein (Merck) were transfected into THP-1 cells using the Neon Transfection System (Thermo Fisher Scientific) according to the manufacturer's instructions (transfection conditions: 1700 V/20 ms/1 pulse). Cell transfection was confirmed by observing GFP presence using an Olympus CKCx5 fluorescence microscope and Countess II Automated cell counter (Thermo Fisher Scientific). Limited dilution cloning was conducted on transfected cells grown in RPMI 1640 medium supplemented with 10% (\(v/v\)) FBS in 96-well plates to establish single-cell-derived colonies. When the cells in each well reached confluency, they were sub-cultured up to T75 flasks, cells were collected, and genomic DNA was extracted using the ISOLATE II Genomic DNA kit (Bioline, Memphis, TN, USA) following the manufacturer's protocol. Polymerase chain reaction (PCR) was conducted to confirm CRISPR/Cas9 cleavage, using two primer sets (primer set 1: 41644-IRAK3_Fwd1 and 41820-IRAK3_Rev bind to sequences flanking the target sites of two sgRNAs; and primer set 2: 41819-IRAK3_Fwd2 binds to a sequence between the target sites of two sgRNAs and 41820-IRAK3_Rev binds to a sequence flanking the target sites of two sgRNAs) given in Supplemental Table S3, with PCR conditions given in Supplemental Table S4.  
The PCR fragments were amplified using the genomic DNA of THP-1 wild type and CRISPR/Cas9-generated cell lines (K5-1 and K6-3) as templates and two primers 41644-IRAK3_Fwd3 and 41645-IRAK3_Rev (Supplemental Table S3) that bind to sequences flanking the target sites of two sgRNAs, using Q5 High-Fidelity DNA Polymerase (New England BioLabs, Ipswich, ME, USA) (initial denaturation at 98 \({}^{\circ}\)C for 30 s, followed by 20 cycles of 98 \({}^{\circ}\)C denaturation for 10 s, 65 \({}^{\circ}\)C annealing for 30 s and an extension at 70 \({}^{\circ}\)C for 2 min; final extension at 72 \({}^{\circ}\)C for 10 min). PCR products were cloned into the vector, pCR-4 Blunt-TOPO of Zero Blunt TOPO PCR Cloning Kit (Thermo FisherScientific) following the manufacturer's protocol. The clones were transformed into One Shot TOP10 competent cells (Thermo Fisher Scientific) and grown in kanamycin(50 \(\upmu\)g/mL)-containing media for selection. Plasmids from 6 to 10 clones were purified using PureLink Quick Plasmid Miniprep Kit (Thermo Fisher Scientific) and sequenced with T3 primer at Micromon Genomics (Monash University). Whole cell protein lysates were subjected to immunoblotting using anti-IRAK3 antibodies and anti-\(\upbeta\)-tubulin antibody for loading control as described in Western Blot section.