GSM5710215: LEPR+ MSC - control [CTR-2]; Mus musculus; RNA-Seq
Long bones were flushed with HBSS supplemented with 2% heat-inactivated FBS, 1% Penicillin/Streptomycin, 1% L-glutamine, 1% HEPES and 200 U/ml Collagenase IV. The flush out was digested for 30 min at 37C.  CD45+ and Ter119+ hematopoietic cells were magnetically depleted. The remaining cells were stained with antibodies against CD45, Ter119, CD31, CD144, LEPR and DAPI. Living CD45- Ter119- CD31- CD144- LEPR+ cells were FACS sorted into DMEM/10% FBS and re-sorted with the same strategy directly into 350 μL RLT plus buffer (Qiagen) with 3.5 μL β-mercaptoethanol. RNA was extracted from the lysates using the RNeasy® Plus Micro Kit (Qiagen) RNA sequencing was performed using the Illumina HiSeq2500 system, with paired-end 2 x 76 bp read length