Mouse lineage depletion kit

Low input libraries were prepared from C57BL6 mice for all normal hematopoietic cell types depicted in Figure 1A. Femurs and tibias were flushed with Hanks Balanced Salt Solution (Gibco) supplemented with 1% heat inactivated Fetal Bovine Serum (HyClone). For lineage depletion, the Mouse Lineage depletion kit (Milteny Biotec 130-090-858, Germany) was used. To preserve RNA integrity, the procedure was carried out at 4°C. Cells were sorted following previously published gating strategies, shown in Figure 1—figure supplement 2 and RNA was extracted using NucleoSpin RNA XS (Machery Nagel, Bethlehem, PA), including DNase treatment. Between 3000 and 10,000 cells were used as library input using Clontech’s SMARTer Ultra Low input RNA Sequencing Kit (Cat. No. 634823, Clontech, Mountain View, CA). cDNA was sheared to 300 bp on a Covaris LE220 and used as input for Clontech’s low input library preparation for Illumina Sequencing kit (Cat. no. 634947). Libraries were sequenced paired-end 100 bp on an Illumina HiSeq2500. Due to the low percentage that HSCs represent in the bone marrow, the libraries were prepared form low numbers of cells as stated above and two biological replicates were produced for each library.

To obtain total bone marrow cells, whole femurs were isolated by removing the attached muscles rapidly. The bone marrow was flushed with PBS using a syringe with a 25-gauge needle. Bone marrow aspirates were subjected to Ficoll Paque gradient centrifugation (Amersham Pharmacia Biotech, Piscataway, USA), and mononuclear cells (MNCs) were separated. MNCs were placed in IMDM (Sigma, USA) containing 20% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin (Gibco, USA). For the experiments, MNCs were incubated in 96-well culture plates (approximately 5 × 10³ BMNCs/well) for 24 to 72 hours. MNCs cells were incubated with media alone or with different concentrations of DMSO (control), rapamycin (100 nM), or bafilomycin A1 (5 nM) for 24 hours at 37 °C. For sorting for hematopoietic stem and progenitor cells, Lineage negative cells were harvested by MACS according to protocol of mouse lineage depletion kit (Miltenyi Biotec, Italy), followed by sorting for HSPCs and HSCs (LSK, and LSKCD34 markers) with FACS (BD Ari III, USA). The subsequent HSPCs or HSCs cells were either analyzed with flow cytometry or transiently cultured in IMDM containing 20% FBS (Hyclon, USA), 100 ng/mL mouse-IL-6, 100 ng/mL mouse Flt-3, 100 ng/mL mouse SCF, 10 ng/mL mouse IL-11 (PeproTech, Rocky Hill, USA).

The bone marrow cells from the animals in the AA groups were obtained on day 14. For sorting of hematopoietic stem and progenitor cells, Lineage negative cells were harvested by MACS according to protocol of a mouse lineage depletion kit (Miltenyi Biotec, Italy), followed by sorting for HSPCs and HSCs (LSK, and LSKCD34 markers) with FACS (BD Ari III, USA). The subsequent HSPCs or HSCs cells were either analyzed with flow cytometry or transiently cultured in IMDM containing 20% FBS (Hyclon, USA), IL-6, Flt-3, SCF, IL-11 (PeproTech, Rocky Hill, USA). The viability of LSKs was assessed using the Presto Blue Assay with 96-well black plates. After 4 μl of PrestoBlue reagent was added to the 96 black plates, the plate was incubated for 30–45 min at 37 °C in an incubator, and the fluorescence was then measured using a Biotek plate reader. After background subtraction, the fluorescence of the sample was divided by the highest fluorescence in the row.