RNAseq of BJ5-ta expressing mScarlet (CTR) and a non-targeting sgRNA
Bulk RNA-seq libraries were assembled from purified mRNA using the Smart-Seq3 workflow94. The reactions were scaled 8-fold per sample, using 15ng input mRNA. First-strand synthesis and template switch reactions were performed by combining RT mix 1 and RT mix 2 and incubated in the thermocycler. cDNA amplification was performed using 20L of the first-strand synthesis reaction as input in the cDNA PCR mix and thermocycled. Amplified cDNA was cleaned up using 0.8x concentration of Ampure XP beads (Beckmen Coulter #A63881). Following clean up, cDNA samples underwent tagmentation and subsequent final library amplification. Briefly, 4ul of tagmentation mix was combined with 400pg of cDNA sample diluted in 4ul of H2O. The tagmentation reaction was incubated at 55 C for 10min, with reaction being stopped with addition of 2L 0.2%SDS. Tagmented samples were then added to the final library mix and amplified. Final libraries were cleaned using 0.8x concentration of Ampure XP beads (Beckmen Coulter #A63881) and quantified using the Agilent Bioanalyzer High Sensitivity DNA (Agilent# 5067-4626) system. Libraries were sequenced on an Illumina NextSeq 500 with paired-end reads at a depth of 10-15 million reads per sample.