pX330-U6-Chimeric_BB-CBh-hSpCas9

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

Vector preparation for the CRISPR/Cas9 system
pX330-U6-Chimeric_BB-CBh-hSpCas9 was purchased from Addgene, (Cambridge, MA, USA). The single guide RNA sequences targeting the Nrp2, Dcstamp, and Nfatc1 eRNA regions were designed using Optimized CRISPR Design39. The guide sequences are listed in Supplementary Table S3. The universal negative control containing a scrambled sequence was 5′-GCACTACCAGAGCTAACTCA-3′40. The targeting vector of each eRNA region was transfected together with the pmaxGFP® vector (Lonza, Basel, Switzerland) into RAW 264.7 cells using the Amaxa Cell Line Nucleofector Kit V (Lonza). The transfected cells were cultured for 2 days, and single-cell sorted using an SH800 cell sorter (Sony, Tokyo, Japan). The knockout clones used in these studies were validated by sequencing.

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Papers

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Manufacturer protocol

Download the product protocol from Addgene for pX330-U6-Chimeric_BB-CBh-hSpCas9 below.

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Videos

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