title: GSM6389851: RBG32105_WT; Mus musculus; RNA-Seq
library_name: GSM6389851
library_construction_protocol: To harvest mouse bone marrow cells, spine, femurs, tibiae and pelvic bones were crushed with a pestle and mortar in 3 mL PBS + 2% FBS. Further 3 mL PBS + 2% FCS were used to wash the bones and the resulting cell suspension was filtered through a 0.75 uM filter. Cells were pelleted and resuspended in 3 mL PBS + 2% FBS. Red cell lysis was performed using cold ammonium chloride (StemCell Technologies cat. no 07800). PBS 2% FCS was added to dilute the red cell lysis buffer during subsequent centrifugation at 4 oC.   For lineage depletion, EasySep™ Mouse Hematopoietic Progenitor Cell Enrichment Kit from StemCell Technologies (cat no. 19816A) was used according to the manufacturer's instructions but avoiding the use of rat serum. Samples were transferred into 5 ml polystyrene (352058) FACS tubes and incubated with 10 µl/ml EasySep mouse hematopoietic progenitor cell isolation cocktail labelled with biotin. 40 µl/ml of EasySep™ Streptavidin RapidSpheres were added and allowed to bind on ice for 10 minutes. The samples were subsequently diluted to 2.5 ml using PBS + 2% FBS and placed in the EasyStep magnet (StemCell Technologies cat no. 18000) for 3 minutes at room temperature. After incubation, samples were transferred into new tubes, spun down, and resuspended in 100 ul PBS + 2% FCS.   For antibody staining, a master mix was prepared as indicated in table 2 and cell suspensions were incubated on ice, in the dark for 30 minutes. After staining, cells were washed, pelleted, and resuspended in 100 ul PBS + 2% FCS. Were necessary, streptavidin conjugated antibody was added in a second step. Samples were pelleted and resuspended to a final volume of 500 ul PBS + 2% FCS. We used single colour, unstained, and full panel controls that were made using pooled BM cells from all mice before lineage depletion. Primary single cells (LSK cells – Lin-Sca1+ckit+) were FACS index sorted into lysis buffer (0.2% Triton X-100 (Sigma), RNase inhibitor (SUPERase, Thermofisher), and nuclease-free water (Thermo Fisher)). scRNA sequencing was performed according to the Smart-Seq2 protocol. Libraries were prepared using the Illumina nextera XT DNA preparation kit and sequenced on the Illumina Hi-Seq 2500.