Fig. 1

Experimental design and RNA-Seq data quality metrics. a Flow chart outlining the experimental design for comparing the three standard input RNA-Seq library preparation protocols. Six xenograft tumors, 3 from the control group and 3 from the experimental group, were used for all three protocols. Similar amounts of tumor tissue from control and experimental groups were used to isolate total RNA. Separate Illumina Stranded Total RNA and mRNA libraries were prepared using 100 ng and 1 μg RNA. The modified NuGEN Ovation v2 protocol library was prepared with 100 ng RNA. Images of the mice and vials were created by the Research Graphics department at MD Anderson Science Park (©MD Anderson), and the pipettes were taken from https://all-free-download.com/free-vectors/ b Flow chart outlining the ultra-low input protocol. Cells from 3 independently derived Zbtb24 wild-type (2lox/+) mESC control lines and 3 independently derived Zbtb24 knockout (1lox/1lox) mESC experimental lines were lysed directly in reaction buffer without isolating total RNA. One hundred cells (~ 1 ng RNA, 18 PCR cycles) and 1000 cells (~ 10 ng RNA, 10 PCR cycles) were used to make cDNA for the TaKaRa SMARTer Low Input RNA-Seq kit v3 protocol. One hundred-fifty pg of TaKaRa SMARTer-generated cDNA was then used to prepare the Nextera libraries. c A diagram depicting the data analysis flow and the data quality metrics used in this study to evaluate RNA-Seq protocols. The analysis steps are on the left and the data quality metrics that were derived from each analysis step are on the right