GSM5923341: CD63+ MSC from WT; Mus musculus; RNA-Seq
For CD11b and HSPC sorting, BM from 1 femur and 1 tibia from one mouse was flushed by centrifugation. For MSC sorting, the rest of the bones (femur, tibia, sternum, hips, humerus, spine) were pooled with all the bones of another mouse and were crushed and digested for 1 hour with collagenase. After erythrocyte lysis, cells were stained with antibody mixes and FACS-sorted using BD Aria II cell sorter. Single cells were encapsulated into emulsion droplets using the Chromium Controller (10x Genomics). Each cell suspension was loaded into one port of a Chromium Next GEM Chip G (10x Genomics) with a target output of 4000-10000 cells. Single cell RNA-Seq libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics) following the manufacturer instructions using a SureCycler 8800 thermal cycler (Agilent Technologies). The average size of each library was calculated using a high sensitivity DNA chip on a 2100 Bioanalyzer (Agilent Technologies) and the concentration was determined using the Qubit fluorometer (Thermo Fisher). Individual libraries were diluted to 10 nM and pooled for sequencing. Library pool was sequenced at 700 pM in paired-end reads (28bp Read1, 10bp Index1, 10 pb Index 2 and 90bp Read2) using one P3 flow cell (100 cycles) on a NextSeq 2000 (Illumina). FastQ files for each sample were obtained using cellranger demux pipeline (10x Genomics). Single cell RNA-Seq libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics) following the manufacturer instructions using a SureCycler 8800 thermal cycler (Agilent Technologies). The average size of each library was calculated using a high sensitivity DNA chip on a 2100 Bioanalyzer (Agilent Technologies) and the concentration was determined using the Qubit fluorometer (Thermo Fisher). Individual libraries were diluted to 10 nM and pooled for sequencing. Library pool was sequenced at 700 pM in paired-end reads (28bp Read1, 10bp Index1, 10 pb Index 2 and 90bp Read2) using one P3 flow cell (100 cycles) on a NextSeq 2000 (Illumina). FastQ files for each sample were obtained using cellranger demux pipeline (10x Genomics). Single-cell RNA-Seq