GSM6286287: 3d7d_SS2_146814.46f [SLX-19981.i727_i507]; Mus musculus; RNA-Seq
All steps in this section (unless otherwise indicated) were performed on ice, and centrifugation steps performed at 300g, 4°C for 5 min. 8-12 weeks old mice carrying the Hoxb5-Cre and the Rosa26-LoxP-STOP-LoxP-tdTomato constructs were treated with 7 daily injections of tamoxifen and sacrificed at indicated time points. Bone marrow cells were extracted from ilia, tibiae and femura by grinding with mortar and pestle in PBS supplemented with 2% Fetal Bovine Serum (cell buffer). The suspension was filtered through a 50µm filter, centrifuged and resuspended in 3 ml of cell buffer. Red blood cells were removed using the ammonium chloride solution: 5 ml of 0.8% Ammonium Chloride (StemCell Tech. 07800) was added to the suspension and incubated for 10 min with intermittent mixing. Afterwards cells were diluted with 7 ml of cell buffer, centrifuged and resuspended in 1 ml of cell buffer. Subsequently, lineage depletion was performed as follows: added 20 µl of the EasySep mouse haematopoietic progenitor cell isolation cocktail (Stem Cell Technologies 19856), incubated for 15 min, added 30 µl magnetic particles, incubated for 10 min, added 1.5 ml of cell buffer and placed tubes in a magnet, incubated for 3 min at room temperature and eluted cells twice (with additional 2.5 ml of cell buffer). Afterwards, cells were centrifuged, resuspended in 200 µl of cell buffer and stained with the antibody panel as follows: antibody mix was added, cells were incubated for 30 min, washed with 2 ml of cell buffer, centrifuged, reuspended in 200 µl cell buffer. For the secondary staining Streptavidin-BV510 was added, cells were washed with 2 ml of cell buffer, centrifuged and resuspended in 1000 µl of cell buffer supplemented with 7AAD. Afterwards cells were sorted with BD influx sorter into either 96 well plates containing 2.3 µl lysis buffer (for the Smart-Seq2 protocol) or 100 µl of PBS with 0.04% BSA in eppendorf tubes ('droplet buffer') when used for the 10x Genomics scRNA-Seq protocol. The Smart-Seq2 plates were vortexed, centrifuged at 800g for 2 min and stored at -80°C. Antibodies and fluorochromes used: Mouse haematopoietic progenitor cell isolation cocktail (Stem Cell Technologies 19856), CD48-APC (Thermo-Fischer 17-0481-82), c-Kit-APC/Cy7 (Biolegend 105826), Sca1-BV421 (Biolegend 108133) and CD150-PE/Cy7 (Biolegend 115914), Streptavidin-BV510 (Biolegend 405234), 7AAD (Thermo Fischer A1310). Smart-Seq2: When cell numbers were limiting single cells were profiled with a modified version of the Smart-Seq2 protocol rather than 10x Genomics kit. Single cells were sorted into 96-well plates with 2.3 µl lysis buffer containing 0.115 µl of SUPERase-In RNase Inhibitor at 20 U/µl concentration (ThermoFisher AM2694) and 0.23 µl of 10% Triton X-100 solution (Sigma 93443), plates were vortexed and stored at -80°C. After thawing 2 µl of the annealing solution (0.1 µ