Nextera XT Library Prep

Prior to using the NEBNext Ultra II FS Library Kit, libraries were prepared using the Nextera XT Kit (Illumina, FC-131-1096). This included the RNA extraction experiments (Fig. ​(Fig.4)4) as well as the AML experiment (Fig. ​(Fig.5B).5B). These libraries were prepared as previously described [11].

Briefly, three replicates of 0.8 ng of DNA were tagmented in 20 μL reactions. Following tagmentation, the libraries were amplified using 0.1 μM P5NextPT5 primer (IDT) and 0.1 μM i7 index primer (IDT) in a reaction volume of 50 μL. The index PCR was incubated as follows: gap fill at 72 °C for 3 min, initial denaturation at 95 °C for 30 s, denaturation at 95 °C for 10 s, annealing at 62 °C for 30 s, elongation at 72 °C for 1 min, and a final elongation at 72 °C for 5 min. Denaturation, annealing, and elongation were repeated for 13 cycles.

Size selection was performed using gel electrophoresis. Libraries were loaded onto a 2% Agarose E-Gel EX (Invitrogen, G401002) and were excised between 300 and 900 bp and cleaned using the Monarch DNA Gel Extraction Kit (NEB, T1020). The libraries were quantified and qualified using an Agilent 2100 Bioanalyzer with a High-Sensitivity DNA analysis kit (Agilent, 5067-4626).