title: GSM6365539: Mesenchymal stem cells, leukemia, rep 1; Mus musculus; RNA-Seq
library_construction_protocol: BM was flushed out of the medullary cavity of femurs and tibias and discarded. The long bones were cut into small fragments with scissors, washed thrice with PBS, and incubated with 1.5mg/ml Collagenase Type 4 (Worthington Biochemical Corp.) and 0.1mg/ml DNase I (Sigma-Aldrich) in PBS supplemented with 10% fetal calf serum (FCS) at 37°C for 60 minutes under agitation. Bone digests were filtered through sterile 100mm cell strainers, and bone fragments were crushed gently using a mortar and pestle in PBS with 2% FCS to detach MSCs. Cells were washed from bone fragments with PBS, strained, and pooled into the bone digests. This was repeated 6 times to maximize the cell yield. Cells were pelleted and treated with Red Blood Cell Lysis Buffer (BD Biosciences), followed by washing and resuspension in 2% FCS/PBS. MSCs were isolated by fluorescent activated cell sorting (FACS). MSCs were identified as CD45-, TER119-, CD31-, Sca-1+, PDGFRa+. Total RNA was extracted from MSCs using the RNeasy Micro Kit (Qiagen). BGI genomics performed RNA sequencing. RNA amplification was performed using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara Bio). Quality control of amplification products was conducted prior to tagmentation-based library construction. Circularization and library quality control was performed prior to sequencing.