Genelute lpa

The filters were subjected to DNA extraction using a homemade protocol with GenEluteTM-LPA (Sigma-Aldrich) solution. The protocol started with a lysis step in Eppendorf tubes by adding 300 μL of TE buffer (TRIS 1M – pH 8, EDTA 0.5M – pH 8) and 200 μL of a lysis solution (TRIS 1M – pH 8, EDTA 0.5M – pH 8 and sucrose 0.7M). Then, a thermic shock was carried out by placing the tubes at −80°C for 15 min and thawed into a block heater at 55°C for 2 min. Next, 50 μL of a 10% sodium dodecyl sulfate (SDS) and 10 μL of proteinase K (20 mg mL^–1) were added to the solution. The solution was incubated at 37°C for 1 h with gentle stirring and placed again in the block heater at 55°C for 20 min. After a quick centrifugation step (13,000 rpm at 4°C for 3 min), the supernatant was collected. Then, 50 μL of sodium acetate (3M – pH 5.2) and 1 μL of GenEluteTM-LPA (Sigma-Aldrich, 25 μg μL^–1) were added. One volume of isopropanol was then added and the tubes were centrifuged for 10 min at 12,000g and 4°C. The supernatant was discarded and 2 washing rounds using ethanol (80%) were carried to purify the DNA. The remaining ethanol was evaporated using a Speed-Vac for 20 min. Finally, 30 μL of TE were added and tubes were left for 1 h at 37°C. DNA concentration was measured using a NanoDrop 1000 spectrophotometer. Afterward, all tubes were stored at −20°C until analysis.

RNA from blood collected in the PAXgene tubes was extracted according to the standard protocol: PAXgene Blood RNA Kit (Qiagen) and stored at −80°C.
RNA quantity and purity was measured using NanoDropTM 1000 Spectrophotometer (Thermo Scientific). OD_260/280 ratios for all samples were between 1.8 and 2.0. RNA integrity number (RIN) was checked using capillary electrophoresis performed on Agilent Bioanalyzer 2100, with RNA 6000 Nano Assay (Agilent Technologies). Sample 1 RIN = 8, sample 2 RIN = 7.3, sample 3 RIN = 7.7, sample 4 RIN = 7.6. Pooled sample 5 for variability modeling had RIN = 7.5.
cDNA synthesis was performed using High Capacity cDNA Reverse Transcription Kit (Life Technology) according to the manufacturer’s protocol with random hexamers in the final volume of 50 μl containing 500 ng total RNA using a cycler C1000 (Bio-Rad). cDNA samples were stored at −20°C and diluted just before use. For dilution of samples GenElute-LPA (Sigma Aldrich) diluted in 1xTE according to the manufacturer instructions was used.

RNA was isolated from FACS purified mouse LK or c-Kit⁺ cells, human CD34⁺ cells from the bone marrow (SCN phase, 1992) or blood (AML phase, 2007) or CD34⁺CD45⁺ HSCs derived from the iPSCs using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s protocol, with the addition of GenElute™−LPA(Sigma-Aldrich). For the in vitro mouse experiments cDNA was generated with the SMARTer Ultra Low Input RNA kit for sequencing (version 3, Clontech), while version 4 of the SMARTer Ultra Low Input RNA kit for sequencing (Clontech) was used for the other samples. Sequencing libraries were generated using TruSeq Nano DNA Sample Preparation kits (Illumina), according to the low sample protocol and run on HiSeq 2500 or Novaseq 6000 instruments (Illumina).