Illumina NovaSeq 6000 paired end sequencing; RNA-seq of RS4:11 cell line populations that are unedited (SPI1 +/+), mono (SPI1 +/-) and biallelicaly edited (SPI1 -/-)
RNA-seq of RS4:11 cell line populations that are unedited (SPI1 +/+), mono (SPI1 +/-) and biallelicaly edited (SPI1 -/-)
RS4:11 cells were electroporated alone (unedited, SPI1+/+) or with CAS9 and a guide RNA sequence (GGGGGUGGAAGUCCCAGUAA) targeting exon three of SPI1 (CRISPR Knockout Pool, Synthego). Editing efficiency approached 50%. Clonal populations of mono (SPI1+/-) and biallelically edited cells (SPI1-/-) were obtained via limiting dilution and clonal expansion. Edits were confirmed via Sanger sequencing and with PU.1 immunoblots.  1,000,000 cells aliquots of SPI1+/+, SPI1+/- and SPI1-/- RS4:11 cells were harvested in in Trizol. RNA was extracted and purified using Direct-zol RNA Microprep (Zymo Research). Approximately 600ng of high-quality RNA was used to prepare each library by using a TruSeq Stranded mRNA Library Prep Kit and the IDT for Illumina-TruSeq RNA UD Indexes (Illumina).