GSM6286647: 3d7d_SS2_146814.46f [SLX-19982.i728_i522]; 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 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 µl of the Maxima enzyme buffer, 0.2 µl of TSO oligo at 100 µM concentration, 1.875 µl of PEG 8000 solution (Sigma P1458) at 40% v/v concentration and 1.275 µl water) and incubation at 42°C for 90 min followed by incubation at 70°C for 15 min. Immediately after, cDNA was amplified by PCR by adding 1 µl of the Terra PCR Direct Polymerase (1.25 U/µl, Takara 639270), 25 µl of the Terra PCR Direct buffer and 1 µl of the ISPCR primer (10 µM stock concentration) to a total volume of 50 µl using the following PCR conditions: 98°C for 3 min, 98°C for 15 s, 65°C for 30 s, 68°C for 4 min (21 cycles), 72°C for 10 min. The amplified cDNA was purified using AMPure XP beads (Beckman A63882), quantified using the PicoGreen assay (ThermoFischer P7589) and used for Nextera library preparation. The libraries were generated using either a standard protocol (batch 7d) or modified protocol (batches 3d7d, 2w4w and 3dr2), see the corresponding metadata) described below. No obvious batch effects were observed among cells analysed with either of the protocols. The standard Nextera protocol: cDNA was diluted to approximately 50-150 pg/µl and 1.25 µl of the solution was used, 2.5 µl of Tagment DNA buffer 1.25 µl of Amplicon Tagment Mix (Nextera XT kit, Illumina FC-131-1096) were added, samples were incubated at 55°C for 10 min, and the reaction was stopped by addition of 1.25 µl of NT buffer. Tagmentation products were amplified by PCR by adding 1.25 µl of each N and S primers (Illumina Nextera XT 96-index kit FC-131-1002) and 3.75 µl of NPM solution and using the following thermocycler settings: 72°C 3 min, 95°C 30 s, 12 cycles of 95°C 30s, 55°C 30s, 72°C 60s and a final extension at 72°C for 5 min. The modified Nextera protocol follows the same principle as the standard Nextera protocol and includes the following steps: cDNA was diluted to approximately 50-150 pg/µl and 1.03 µl of the solution was used, 1.63 µl of Tagment DNA buffer and 0.6 µl Amplicon Tagment Mix was added, samples were incubated at 55°C for 10 min, the reaction was stopped by adding 0.82 µl of NT buffer. Tagmentation products were amplified by adding 1.23 µl of each N and S primers (as above but diluted 5 times), 2.3 µl of Phusion HF buffer (ThermoFischer F530L), 0.1 µl of dNTP (25 mM stock concentration), 0.07 µl of Phusion polymerase and 2.5 µl of water and using the following thermocycler settings: 72°C 3 min, 98°C 3 min s, 12 cycles of 98°C 10s, 55°C 30s, 72°C 30s and a final extension at 72°C for 5 min. Libraries were sequenced using the Illumina Hiseq4000 or NovaSeq instruments, obtaining approximately 100-200 mln reads per 96 cells. 10X genomics: For the 10x Genomics scRNA-Seq protocol up to 20,000 cells were pooled in pairs corresponding to male and female animals, centrifuged and resuspended in a volume of droplet buffer optimal for recovery of up to 10,000 cells and immediately processed with the 10x Genomics Single Cell 3' v3 protocol following the manufacturer's instructions. Libraries were sequenced using the Illumina NovaSeq instrument, obtaining at least 20,000 reads per cells.