GSM9498239: GFPscreen_GFP+tronslons_replicate2; Mus musculus; OTHER
Following recovery cells were trypsinized and resuspended in 1mL PBS+ 1% FBS to reduce clumping. 100µL of the cells were stored as input and remaining 900µL cells were passed through a cell strainer to achieve single cell suspension and immediately sorted using a Sony MA900 FACS machine using the maximum purity settings. Singlets were gated using a SSC-H vs SSC-W plot followed by sequential gating using a FSC-A vs FSC-H plot. Singlet cells showing a 10 fold increase in GFP were considered GFP positive and sorted in PBS+ 5% BSA to increase centrifugation efficiency. Sorted cells were centrifuged and the pellets were used for genomic DNA isolation using Zymo mini DNA isolation kit. The 5′UTR sequences upstream of the nostartGFP were amplified from 2 µg gDNA in a 100µL PCR reaction divided into four 25 µL reactions for 22 cycles using custom made Illumina indexing primers to generate amplicon libraries (Supplemental table S8). PCR products were cleaned with AmpureXP beads and quantified on a bioanalyzer. Guide amplicon libraries were sequenced using NovaSeq 6000 (Illumina) with a PE150 chemistry.