Bamhi hf

pT3TS-nCas9n (a gift from Wenbiao Chen: Addgene plasmid # 46757) was linearized with XbaI (NEB R0145. Ipswich, MA), whereas TALEN constructs were linearized with SacI-HF (NEB R3156. Ipswich, MA) and sgRNA vector with BamHI-HF (NEB R3136. Ipswich, MA). Tyr sgRNA #2 –a construct made in the Essner Lab–was linearized with HindIII (NEB R0104. Ipswich, MA). RNA was made using T3 mMessage mMachine kit (Ambion AM1348. Foster City, CA) or HiScribe T7 High Yield RNA synthesis kit (NEB E2040. Ipswich, MA) according to manufacturer’s protocols with the addition of RNA Secure to the reaction (Ambion AM7010. Foster City, CA). To purify RNA, phenol-chloroform extraction was performed using Acid Phenol, Chloroform, and MaXtract High Density Tubes (Qiagen 129046. Hilden, Germany). RNA was then precipitated with Isopropanol at -20 °C, pelleted, air dried and resuspended into nuclease free water. The quality and quantity of RNA were ascertained by using a Nanodrop spectrophotometer and running aliquot on agarose gel. Each batch of RNA was aliquoted into small single use tubes and stored at -80 °C until the morning of microinjections.

The manipulation of genetic material and the generation of genetically modified organisms was approved by the UTS Biosafety Committee (2001-19-R-GC; 2009-02-R-GC). Briefly, the luciferase reporter gene Luc2 (Photinus pyralis), encoded within the vector pGL4.20 (Luc2/Puro) (Promega, USA), was digested with the restriction enzymes EcoRV-HF and BamHI-HF (New England Biolabs, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro-mcs (InvivoGen, USA), to generate the plasmid pVITRO2-Luc2. The CDUPRT gene encoded by the plasmid pORF5-Fcy::Fur (InvivoGen, USA), was digested with EcoRI-HF and NheI-HF (New England Biolabs, USA), and ligated into the pVITRO2-Luc2 plasmid, to generate the plasmid pVITRO2-Luc2/CDUPRT.

In order to amplify the knockout fragment containing approximately 700 bp upstream and downstream region of targeted gene, flanking primers with 15-18 bp overlap (Table S1) were designed. The upstream and downstream regions were first amplified using the high-fidelity Platinum SuperFi PCR master mix (Invitrogen) and purified from a 1% agarose gel (QiAquick gel extraction kit, Qiagen). The fragments were then spliced together by overlap extension PCR and gel purified. Plasmids were isolated from overnight cultures using Wizard Plus SV miniprep kit (Promega). Plasmid and the knockout fragment were digested using SpeI-HF or BamHI-HF (New England Biolabs) for 2 h at 37 °C and gel purified. The restriction digested knockout fragment and plasmid were ligated together using T4 DNA ligase (New England Biolabs) and transformed into E. coli Jke201 using the heat-shock method. After a heat shock, the cells were resuspended in pre-warmed LB broth supplemented with 100µM DAP and incubated at 37 °C for 2 h before plating on an ampicillin (100 µg/ml) selection plate. Transformants were screened by colony PCR using the primers flanking the upstream and downstream regions of the targeted gene (Table S1).

Single clones were inoculated in 25 mL LB Medium containing 0.1 mM Kanamycin and incubated overnight at 37 °C and 200 rpm. Plasmid DNA was isolated using Presto™ Mini Plasmid Kit (Geneaid Biotech Ltd., New Taipei City, Taiwan). A total of 1 µg of plasmid DNA, isolated from overnight cultures of selected single clones, was restriction digested with BamHI-HF (New England Biolabs (NEB), Ipswich, MA, USA). Correct size of digestion products was verified by gel electrophoresis using MetSV genomic DNA isolated from lysed M. mazei cultures by ultracentrifugation at 126,100× g for 4 h at 4 °C followed by processing using QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany), according to manufacture protocol. Three candidate plasmids were selected (pRS1626, pRS1782 and pRS1783) and stored in E. coli glycerol cultures at −80 °C, named K4679, K4871 and K4872.

Barcodes were synthesized as oligonucleotides from IDT (Coralville, IA), and are listed in the electronic supplementary material, table S3 as ClonalBarcode5 and ClonalBarcode3. The oligonucleotides were annealed and ligated into pBabe Puro (Addgene #1764, Addgene, Cambridge, MA) that had been digested with BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs), treated with calf intestinal phosphatase (NEB), and purified with a PCR purification kit (Qiagen). One microlitre of 150 nM annealed clonal barcode was ligated to 190 ng of digested pBabe Puro using T4 ligase (New England Biolabs) overnight at 16°. The ligation product was purified using 1× volume of AMPureXP beads (Beckman Coulter) as per the manufacturer's protocol, and eluted into 20 µl. Four times, 2 µl of purified ligation product was transformed into 40 µl of DH5α Electromax Escherichia coli (Fisher Scientific). Transformed bacteria were allowed to recover in 1 ml SOC medium, pooled and plated on LB Agar with 100 µg ml⁻¹ Ampicillin in two 245 mm plates (Corning, Corning, NY). Some transformed mixture was diluted and plated for counting and colony estimation; this yielded an estimate of approximately 1.7 × 10⁶ colonies. After overnight growth at 37°C, colonies were scraped off and plasmid DNA was extracted with a Gigaprep kit (Qiagen).

cDNAs of ASCC1, ASCC2, ASCC3, and cGAS were bought from Horizon, were amplified with primers adding attB1 and attB2 sequences (Table S3) and were cloned into the pDONR223 vector using the gateway BP recombinase system (Thermo Fisher Scientific, 11789020). All cGAS mutants were generated using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, E0554S) with specific primers (Table S3) and verified by sequencing. pDONR223 constructs were recombined into the pFRT/ TO/FLAG/HA-DEST or pFRT/TO/GFP-DEST destination vector using Gateway LR Clonase II Enzyme mix according to the manufacturer’s protocol (Thermo Fisher Scientific, 11791020). pGEX6p-1 cGAS WT was generated using In-Fusion® HD Cloning Kit (Takara Bio USA, 102518). pGEX6p-1 cGAS 8his (c-terminus) was generated using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, E0554S). pGEX6p-1 hPrimpol1 was generated using BamHI-HF (New England Biolabs, R3136S) and NotI-HF (New England Biolabs, R3189S) restriction enzymes