Ssofast sybr green

Cells were sorted from mouse bone marrow as described above and analyzed for gene expression as performed previously (Reynaud et al., 2011 (link); Pietras et al., 2015 (link); Pietras et al., 2016 (link); Hernandez et al., 2020 (link); Rabe et al., 2020 (link); Chavez et al., 2021 (link); Ahmed et al., 2022 (link); Chavez et al., 2022 (link)). Cells were sorted at 100 cells per well in 5 μL CellsDirect reaction buffer (Invitrogen). RNA was then reverse transcribed and preamplified with a panel of 96 DeltaGene Assay primer sets (Fluidigm) for 20 rounds with Superscript III (Invitrogen) and subsequently treated with Exonuclease I (New England Biolabs) to remove non-target genetic material. cDNA was then diluted in DNA suspension buffer and loaded onto Fluidigm 96.96 Dynamic Array IFCs along with the DeltaGene Assay primers and run on a Biomark HD (Fluidigm) using SsoFast Sybr Green (Bio-Rad) as a detector. Data were analyzed using the ΔΔCT method and normalized to Gusb expression. Hierarchical clustering and PCA analyses were performed using ClustVis. As Gusb was used to normalize data, it was not included in clustering and PCA analysis. A complete list of all Fluidigm primer sequences can be found in Supplementary Table S4. Hoxa2 and Ebf1 were excluded from all analyses due to poor primer performance in our studies.

Fluidigm qRT-PCR analysis was performed as previously described [7 (link)]. Briefly, 100 PU.1 low and PU.1 high SLAM cells per well were sorted directly into 5 µL of 2× Reaction Mix (Invitrogen, Waltham, MA, USA) contained within a 96-well PCR plate. Once all cells were sorted, the plates were sealed with an aluminum seal, spun in a centrifuge at 1200 RPM for 5 min, snap frozen in liquid nitrogen, and stored at −80 °C. cDNA was generated from RNA using Superscript III (Invitrogen) and a custom primer set mix (Fluidigm, San Francisco, CA, USA) that were amplified for 18 cycles on a thermocycler (Eppendorf, Hamburg, Germany). Excess primers were removed from the samples by Exonuclease I (NEB, Ipswich, MA, USA) treatment, and the samples were diluted in DNA suspension buffer (Teknova, Hollister, CA, USA). Pre-amplified cDNA and custom primer sets were loaded onto a Fluidigm 96.96 Dynamic Gene Expression IFC. Subsequently, the IFC was run on a Biomark HD (Fluidigm) with SsoFast Sybr Green (Bio-Rad, Hercules, CA, USA) used for detection. Fluidigm gene expression software was used to analyze the data, and all values are relative to Gusb. Relative changes in gene expression were determined using the ΔΔCT approach.

RNA was isolated from flash-frozen cell pellets and tumour tissue (GenElute: Sigma-Aldrich, USA) and cDNA was generated with 500 ng RNA using a reverse transcription kit (Bio-Rad iScript: Qiagen, USA). RNA quantity and quality were assessed with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). All QPCRs were run on an ABI 7500 FAST machine (Applied Biosystems, USA) with a commercial mastermix (TAqMan: Applied Biosystems, USA; RT² SYBR green: Qiagen, USA; or SsoFAST SYBR green: BioRad, USA). QPCR primers (Table S1) amplified single products of the expected size with a single-peak melt curve. Primers were further validated to ensure that primer efficiency was close to 100% and closely matched efficiency of housekeeping genes, with a dynamic range of at least three orders of magnitude. The QPCR-based RT² profiler array was performed similarly, following the manufacturer’s protocol (Qiagen, USA).