NEOold_KO_3
Mus musculus
Protocols: Bone marrow was isolated from hindlegs (femur, tibia), hips (ilium) and backbone (vertebra). Muscle, connective tissue and spinal cord were removed, bones were crushed in RPMI/ 2% FBS (Gibco, Carlsbad, CA) using mortar and pestle. Single cell-suspensions were made by flushing through a 40 m filter mesh. Cell numbers and viability were determined using a ViCell Counter (Beckman Coulter, Brea, CA). To deplete lineage-positive cells we used the Dynabeads Untouched Mouse CD4 Cells Kit (Invitrogen). Briefly, total BM was stained for 30 min with a 1:5 dilution of the Lineage Cocktail provided in the Dynabeads Untouched Mouse CD4 Cells Kit (Invitrogen). Labeled cells were then incubated for 20 min with 1.5 mL/mouse of washed polyclonal sheep anti-rat IgG coated Dynabeads provided in the Kit. Cells were depleted using a magnet, enriching for the lineage-negative (Lineage-) cell fraction. To purify HSC and MPP1-4, the Lineage- fraction was stained for 30 min using the following monoclonal antibodies (all eBioscience): anti-lineage [anti-CD4 (clone GK1.5), anti-CD8a (53-6.7), anti-CD11b (M1/70), anti- B220 (RA3-6B2), anti-GR1 (RB6-8C5) and anti-TER119 (Ter-119)]; anti-CD117/c-Kit (2B8); anti-Ly6a/Sca-1 (D7); anti-CD34 (RAM34); anti-CD150 (TC15-12F12.2); anti-CD48 (HM48-1); anti-CD135 (A2F10); CD45.1 (A20); CD45.2 (104). The coupled fluorochromes depended on the experiment to allow sorting of different fluorescent protein containing reporters or isolation of HSC from transplants. Monoclonal antibody conjugates were purchased from eBioscience or BioLegend. Cell sorting was then performed on a FACS Aria I, II and III or FACS Aria Fusion (Becton Dickinson) directly into lysis buffer. Total RNA isolation was performed from the indicated populations using ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen) according to the manufacturers instructions. DNAse treatment was performed using RNAse-free DNAse Set (Qiagen). Total RNA was used for quality controls and for normalization of starting material cDNA-libraries were generated with 1 ng of total RNA using the SMARTerTM Ultra Low RNA Kit for Illumina Sequencing (Clontech) according to the manufacturers indications. Of note, twelve cycles were used for the amplification of cDNA respectively. Paired-end adaptors were applied to each population.