GSM4859443: post-TAC HSC rep1 [RNA-seq]; Mus musculus; RNA-Seq
Bone marrow cells were harvested from tibias, femurs, pelvis, and vertebrae by crushing with a mortar and pestle in MACS buffer (PBS supplemented with 0.5% bovine serum albumin and 2 mM EDTA). The cells were passed through a 25G needle several times and filtered with a 70 μm cell strainer. After washed and filtered with a 40 μm cell strainer, the samples were incubated with Mouse CD117 Microbeads (Miltenyi) and separated by autoMACS (Miltenyi). Isolated cells in positive selection were mixed with antibodies for flow cytometry. CD45+ Lin- Sca1+cKit+CD34-Flt3-CD150+CD48- cells were sorted as HSCs. Sorted cells were lysed in Buffer RLT plus (QIAGEN) and RNA was harvested using RNeasy micro Kit (QIAGEN). Poly-A mRNA was extracted from total RNA by using an Oligo-dT beads of NEBNext Poly(A) RNA Magnetic Isolation Module (NEB), after which RNA-seq libraries were prepared using a NEBNext Ultra II RNA Library Preo Kit for Illumina (NEB) according to the manufacturer's protocol.