Spliced 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. Two weeks after library integration (and before Cas9 treatment), RNA (Qiagen RNEasy Maxi kit) was 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.