RNA-Seq Analysis of FACS-sorted GFP-expressing and skeletal stem cells (SSC) from the bone marrow of mice with S1P ablation in the Osterix (Osx)-lineage (Cko)

Comparison of the transcriptome of GFP-expressing cells from the bone marrow compartment of Cko mice with the transcriptome of skeletal stem cells (SSC) from the bone marrow compartment of Cko and WT mice (homozygous floxed S1P mice). Forelimbs and hindlimbs from mice were crushed in a mortar and pestle in the presence of phosphate buffered saline (PBS) and strained through a 70 micron filter and filtrate stored on ice. Crushed bones were digested with collagenase at 37 degree celcius for 15 min, strained through a 70 micron filter and pooled with the filtrate from above. The filtrate/cells were washed with cold PBS, and the RBCs lysed using an RBC lysis buffer (Sigma, R 7757) for 5 min. Following washings with cold PBS to remove lysed cells and hemoglobin, cells were suspended in PBS with 0.1% bovine serum albumin and stained with fluorescence-conjugated CD45, Ter-119, CD31, and CD105 antibodies and FACS-sorted for skeletal stem cells (SSC) defined as the population of cells positive for CD105 antigen but triple negative for Ter-119, CD31, and CD45 markers. Single cells were collected directly into 96 well plates from two WT (WT3 and WT5) and two Cko (Cko2 and Cko4) individually, lysed and RNAseq performed from RNA harvested from each well. Similarly, single GFP-expressing cells were directly FACS-sorted (i.e. without the use of any fluorescence conjugated antibodies) from the pooled bone marrow compartment of two Cko mice (in duplicate).本研究数据集对比了条件性敲除（conditional knockout, Cko）小鼠骨髓腔中表达绿色荧光蛋白（green fluorescent protein, GFP）的细胞转录组，与纯合S1P基因floxed的Cko及野生型（wild type, WT）小鼠骨髓腔中骨骼干细胞（skeletal stem cells, SSC）的转录组。具体实验流程如下：1. 取小鼠前后肢，置于磷酸盐缓冲液（phosphate buffered saline, PBS）中，经研钵与研杵粉碎后，通过70μm滤网过滤，滤液置于冰上保存。2. 将粉碎的骨组织于37℃下用胶原酶消化15分钟，经70μm滤网过滤后，与上述滤液合并。3. 合并后的滤液与细胞用预冷PBS洗涤，随后使用红细胞裂解缓冲液（Sigma, R 7757）裂解红细胞5分钟。4. 经预冷PBS洗涤以去除裂解细胞与血红蛋白后，将细胞重悬于含0.1%牛血清白蛋白的PBS中，采用荧光标记的CD45、Ter-119、CD31及CD105抗体进行染色，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分选出骨骼干细胞（SSC）：该细胞群体需满足CD105抗原阳性，同时Ter-119、CD31与CD45标记均为阴性。5. 分别从2只编号为WT3、WT5的WT小鼠，以及2只编号为Cko2、Cko4的Cko小鼠中，直接将单细胞收集至96孔板，裂解后提取每孔中的RNA并开展RNA测序（RNA sequencing, RNAseq）。6. 此外，从2只Cko小鼠的混合骨髓腔中设置生物学重复，直接通过FACS分选（无需使用荧光标记抗体）得到表达GFP的单细胞。