Deoxyribonuclease 1

Mouse embryonic fibroblasts (MEFs, isolated at E14.5) were prepared as previously described [5] (link). Briefly, embryos were harvested from mice of mixed background at 14.5 dpc followed by decapitation and removal of internal visceral organs, including the heart. The tissue was minced and digested with trypsin and trituration. Cells were resuspended in MEF medium (10% FBS and 2 mM L-Glutamine) and plated onto one 10 cm dish per embryo. After 24 hours, cells were passaged at 1∶3 (passage 1). MEFs were used at passages 3–5 for all reprogramming experiments.
Adult mouse cardiac fibroblasts were prepared as previously described [5] (link). Hearts were removed from mice (8–12 weeks in age) and minced in cold PBS. The tissue was digested in 4 mg mL⁻¹ collagenase IV (Sigma) and 10 U mL⁻¹ deoxyribonuclease I (Worthington Biochemical Corporation) and agitation at 37°C for 10 minutes. Samples were spun down and resuspended in TrypLE (Invitrogen) at 37°C with agitation. After 5 minutes, medium (DMEM supplemented with 15% FBS, 1% NEAA) was added and the resulting solution was plated onto gelatin coated 6-well plates. When confluent, the cells were passaged after filtration through a 40 µM filter at 1∶1 to a gelatin coated 10 cm dishes (passage 1). Cells were then passaged 1∶5 and frozen when confluent. Cardiac fibroblasts were used at passage 3 for reprogramming experiments.

As previously described (Stanley et al., 2008 (link)) mice were infected with L. donovani and sacrificed 5 hours later. Spleens were digested in collagenase type IV (1 mg/ml; Worthington, Lakewood, NJ) and deoxyribonuclease I (0.5 mg/ml; Worthington) at RT for 45 minutes. Splenocytes were processed as described above. Splenic DCs were isolated from splenocyte preparations by positive selection with anti-CD11c MACS beads, according to the manufacturer’s instructions (Miltenyi Biotec). DCs were also stained for MHCII and CD86 and analyzed by flow cytometry, as described above. Purified DCs were stored in buffer RLT and homogenized in QIAshredder columns (both from QIAGEN). Total RNA was extracted from purified DC using the RNeasy Mini Kit with on-column DNase digestion (both from QIAGEN). RNA samples were reverse transcribed into cDNA using the cDNA Archive Kit (Applied Biosystems, Foster City, CA). RT-qPCR for Il12 and Il10 was performed on CF384 Touch Real-Time PRC Detection System (BIO-RAD) using the TaqMan Gene Expression Assay (Applied Bioscience). Relative quantification was performed using the comparative C_T method relative to Hprt and beta2m.

Skin biopsies (1 6 mm biopsy at 6 hr and 5 6 mm biopsies at 24 hr) from female B6 and cGAS−/− mice were obtained prior to, 6 hr, and 24 hr after skin exposure to UVB light. Skin biopsies were processed as previously described^{13 (link)}. Briefly, the tissue was minced finely and digested with 0.28 units/ml Liberase TM (Roche) and 0.1 mg/ml of Deoxyribonuclease I (Worthington) in PBS with Ca⁺² and Mg⁺² for 60 minutes at 37 °C with shaking. Cells were passed through a 0.7 μm strainer, resuspended in RPMI and counted. Cells were treated with Fc Block TruStain FcX (Biolegend) and stained in PBS + 1% BSA. Surface staining was performed using mouse–specific fluorescent antibodies purchased from Biolegend: i) Myeloid panel: CD45 APC.Cy7, CD11b PerCP.Cy5.5, Ly6C Violet 610, Ly6G FITC and ii) T cell panel: CD45 APC.Cy7, CD8 PerCP.Cy5.5, CD4 Pe.Cy7, γδ T PE. Samples were processed using CytoFLEX flow cytometer (Beckman Coulter) and data analyzed with FlowJo software v10 (Tree Star). Flow cytometry gating is demonstrated in Supplementary Figure S6. Immune cell populations and mean fluorescence intensities (MFI) of Sca-1 were determined using fluorescence minus one (FMO) controls. Numbers of different cell populations were determined based on total counted cell numbers and normalized to the area of a single biopsy (6 mm).

A549 cells prepared in six 15-cm dishes (approximately 1.0 x 10⁸ cells in total) were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4°C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer (+) supplemented with halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) and 5 μg/ml of deoxyribonuclease I (Worthington Biochemical Corporation, Lakewood, NJ). Lysate was clarified by centrifugation at 3,900 x g at 4°C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin (QIAGEN) at 4°C. After 2 h of incubation, the resin was washed three times with PD lysis buffer (+) and once with PD lysis buffer without TritonX-100 (PD lysis buffer [–]). After the centrifugation at 1,600 x g and 4°C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer (-) containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion.