pSpCas9(BB)-2A-GFP (PX458)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Publication protocol

RAW264.7 KO cell lines and complementation
All RAW264.7 KO cell lines were created using the CRISPR/Cas9 system (Cong et al., 2013). Target gRNA sequences were generated using CRISPR Design and are listed in Table S2. The target gRNAs were then cloned into pX458 following a previously published protocol (Cong et al., 2013). A total of 106 RAW264.7 macrophages were transfected with 3 µg pX458 in a 6-well tissue culture treated plate with 6 µl Fugene (Promega). 48 h pt, macrophages were single-cell sorted into 96-well tissue culture–treated plates with 200 µl of DMEM + 10% FBS. After 2 wk of incubation, gDNA from macrophages colonies was purified using QIAamp DNA mini kit (QIAGEN). The targeted DNA sequence was then amplified and sequenced (Elim Biopharm) with primers (Table S2).

Retroviral constructs were transduced into RAW264.7 macrophages using vesicular stomatitis pseudotyped virus packaged in 293T cells and selected for 48 h with 5 µg/ml puromycin (InvivoGen). Caspase-11 and Cpb1 were cloned in-frame into pMSCV-FLAG-HA-IRES-Puro using Gateway Cloning, as previously described (Storek et al., 2015).



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Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-GFP (PX458) below.

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