Primary transcript sequencing of mus musculus: embryonic stem cells
Our library of splice acceptor sequences targeted by multiple gRNAs was constructed through a multistep process, cloned into a plasmid backbone allowing Tol2 transposon-based integration into the genome, and integrated into the genomes of mESCs using Lipofectamine 3000 transfection along with equal molar Tol2 transposase followed by one week of Hygromycin selection to ensure genomic integration. One week after library integration, the mESCs were transfected with p2T CAG Cas9 BlastR (Addgene 107190) and Tol2 transposase using Lipofectamine 3000 followed by one week of Blasticidin selection to maximize Cas9 activity. RNA (Qiagen RNEasy Maxi kit) was then extracted from separate aliquots of each replicate culture. Samples were prepared for Illumina Nextseq using RT-PCR based methods, and paired-end high-throughput DNA sequencing (Illumina Nextseq 2 x 75-nt kit) was performed to yield this dataset.