Supplementary Figure 1: Cloning and validation of the CROPseq-Guide-Puro plasmid
From: Pooled CRISPR screening with single-cell transcriptome readout
a) As starting point for preparing the CROPseq-Guide-Puro plasmid, we amplified four PCR products (A, B, C, and D) from the LentiGuide-Puro plasmid with the indicated primer pairs. b) CROPseq-Guide-Puro was constructed from these four amplicons using the ligase cycling reaction (LCR). The assembly was directed by four overlapping bridge oligonucleotides to flip the order of parts C and D. This rearrangement places the hU6-gRNA cassette into the 3′ LTR, downstream of the EF-1a puromycin marker. c) To validate the duplication of the hU6-gRNA cassette during lentiviral integration, we performed PCRs with primers that bind to the hU6 promoter but face in opposite directions. Productive amplification can occur only when amplifying from a circular plasmid or following duplication of the cassette during viral integration. As templates, we used gDNA from LentiGuide-Puro transduced cells (lane 1, resulting in no amplification), a plasmid preparation of CROPseq-Guide-Puro (lane 2), or gDNA from CROPseq-Guide-Puro transduced cells (lane 3).
