Idt duplex buffer

Antisense oligonucleotides (DNA-PS and 2′ OMe-PS gapmers), primers and probes were chemically synthesized using standard phosphoramidite chemistry. RNA oligonucleotides (21mer unmodified siRNAs and 27mer DsiRNAs) were chemically synthesized using t-Butyl-dimethylsilyl (TBDMS) chemistry (Integrated DNA Technologies). Probes for qPCR were purified using reversed phase high performance liquid chromatography (RP-HPLC) (Integrated DNA Technologies) while all other oligonucleotides were prepared as sodium salts. All oligonucleotides were analyzed by electrospray-ionization mass spectrometry (ESI-MS) and were within ±0.02% predicted mass. Oligonucleotide concentrations were calculated using modification-specific extinction coefficients based on measured ultraviolet (UV) absorbance at 260 nm. RNA duplexes were annealed in IDT duplex buffer (30 mM HEPES, pH 7.5, 100 mM Potassium Acetate) (Integrated DNA Technologies). Silencer^® Select siRNAs were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and LNA™ longRNA GapmeRs were purchased from Exiqon (Vedbaek, Denmark). All oligonucleotide sequences are shown in Supplementary Table S1 in the online Supplemental Data.

Small hairpin RNAs targeting RUNX1, CBFβ and ETV6-RUNX1 were designed using several prediction tools or The RNA Consortium (TRC) website (please refer to Supplementary Table 5 for exact sequences). In general, a sequence of 21 nt in length starting with G was chosen where possible. The loop sequence (GGGATCCG) was designed to contain the BamHI (GGATCC) restriction site, not present in the LLX3.7 vector, which was then used for selection of positive clones. Sequences were ordered as primers from Integrated DNA Technologies (IDT) website and resuspended in DNase–free water as a 100 μM stock concentration. For oligo annealing 1 μl of 100 μM forward and reverse primers for each shRNA construct were mixed in a PCR tube with 48 μl of IDT Duplex Buffer (Integrated DNA Technologies) and cloned into the LLX3.7 lentiviral vector. For TRIPZ vector shRNA were designed as in Fellmann et al.^{56 (link)} and cloned following manufacturer’s instructions. To induce knockdown doxycycline was added at a final concentration of 0.5 μg/ml and cells were collected for western blot analysis 72 h later. For RNA-seq cells were collected 7 days post induction with Doxycycline.

Cas9 gRNAs were prepared by mixing equimolar amounts of Alt-R™ crRNA and Alt-R tracrRNA (Integrated DNA Technologies, Coralville, IA, USA) in IDT Duplex Buffer (30 mM HEPES, pH 7.5, 100 mM potassium acetate; Integrated DNA Technologies), heating to 95 °C and slowly cooling to room temperature or using Alt-R sgRNA (Integrated DNA Technologies) hydrated in IDTE pH 7.5 (10 mM Tris, pH 7.5, 0.1 mM EDTA; Integrated DNA Technologies). Cas12a gRNAs consisted of Alt-R Cas12a crRNAs (Integrated DNA Technologies) hydrated in IDTE pH 7.5. RNP complexes were assembled by combining the CRISPR–Cas nuclease (Alt-R S.p. Cas9 Nuclease V3, Alt-R S.p. HiFi Cas9 Nuclease V3, Alt-R S.p. Cas9 D10A V3, Alt-R S.p. Cas9 H840A V3, Alt-R A.s. Cas12a V3, or Alt-R A.s. Cas12a Ultra; Integrated DNA Technologies) and the Alt-R gRNA at a 1:1 to 1.2:1 molar ratio of gRNA:protein and incubating at room temperature for 30 min. For paired nicking experiments, each RNP was formed separately, and two RNPs were mixed together at an equal molar ratio prior to adding to the cells at the time of transfection. The 20-nt target specific sequences of the gRNAs used in this study are listed in Supplementary Table 1.