GSM7372533: Mouse1_LTHSCs_Hoxb5_mKOneg [SLX.17004.i726_i505]; Mus musculus; RNA-Seq
All steps in this section (unless otherwise indicated) were performed on ice, and centrifugation steps performed at 500g, 4°C for 5 min. 8-12 weeks old mice carrying the Hoxb5-mKO2 reporter were sacrificed and cells were isolated from bone marrow (femurs and tibia) 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 50ul of FC block, incubated for 5 min and 50ul of antibody cocktail was added. The antibody cocktail contained DAPI and the following antibodies: CD48-APC, CD34-FITC, CD135-PE/Cy5, CD150-PE/Cy7, Lin-biotin, Sca1-APC/Cy7, cKit-BV711. ml of cell buffer. The suspension was incubated for 20min on ice, washed with 1ml of cell bufffer, centrifuged and resuspended in 100ul of Streptavidin-PB, incubated for 15 min on ice, washed with 1ml cell buffer, centrifuged and resuspended in 300ul of cell buffer with addition of DAPI. Cell suspension was filtered as above and sorted with BD influx sorter into 96-well plates containing 2.3 µl lysis buffer (for the Smart-Seq2 protocol). The Smart-Seq2 plates were vortexed, centrifuged at 800g for 2 min and stored at -80°C. 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 µl of ERCC RNA Spike-In solution (1:300,000 dilution) Thermo-Fisher 4456740), 0.02 µl of the oligo-dT primer (100 µM stock concentration) and 1 µl of dNTP (10 mM stock concentration)) was added. The plate was incubated at 72°C for 3 min, cooled down on ice and reverse transcription was performed by adding 5.7 µl of RT buffer (0.1 µl of Maxima H minus reverse transcriptase at 200 U/µl concentration (ThermoFischer EP0752), 0.25 µl of SUPERase-In RNAse Inhibitor at 20 U/µl concentration, 2 µ