title: RNA-seq of homo sapiens: MCF10A ERK DS cells + TRE3G::BRAF-V600E, 24hr dox treatment
design: RNA-seq sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (E7770S, NEB) in combination with the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490S, NEB). PolyA tail selection and library preparation were performed in the same batches as extraction in order to reduce batch effects. Briefly, approximately 1 ug of total RNA was used as input to polyA tail selection for each sample. After polyA selection, samples were fragmented for 10 minutes at 94C to achieve RNA fragmentation between 200-500 bp, followed by first and second strand cDNA synthesis. DNA end repair and adapter ligation were then performed and followed by size selection according to manufacturers recommendations to achieve a final insertion size of ~300 base pairs (bp) and final library size of ~420 bp. Samples were amplified following manufacturers recommendations for 1 ug of input using unique combinations of i5 and i7 primers from NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 (E7600S, NEB). Combinations of i5 and i7 primers were chosen by using the checkMyIndex tool with settings for NovaSeq chemistry. Libraries were quantified using Qubit dsDNA Broad Range assays (Q32850, Thermo Fisher Scientific). Library size was determined using a Genomic DNA ScreenTape in a TapeStation 4200 (G2991BA, Agilent). Completed libraries were stored at -20C until sequencing. Libraries were diluted to 4 nM, pooled at equal ratios, and 2x150 bp paired-end sequencing was performed on two lanes of a SP flow cell (300 cycle) on an lllumina NovaSeq 6000 instrument. RNA-seq NovaSeq 6000 sequencing was conducted at the Genetic Resources Core Facility (RRID: SCR_018669), Johns Hopkins Department of Genetic Medicine, Baltimore, MD.