GSM6856766: RLT10; Mus musculus; RNA-Seq
Long bones were flushed with HBSS supplemented with 2% of heat-inactivated fetal bovine serum, Penicillin/Streptomycin, L-glutamine, HEPES, and 200 U/mL Collagenase IV (Worthington Biochemical Corporation) and digested for 30 min at 37°C. Cell clumps were dissociated by gentle pipetting. Cells were filtered through a 100 mm nylon mesh and washed with HBSS/2% FBS. Hematopoietic cells were depleted by staining with biotin-conjugated CD45 and Ter119 antibodies, and using Dynabeads ® Biotin Binder (Invitrogen #11047). Following depletion of hematopoietic cells, the remaining cells were stained with antibodies against CD31, CD144 and PDGFRb. BM MSCs were identified as CD45- Ter119- CD31- CD144- PDGFRb+ cells. Sorting was performed using a BD FACS Aria II. Cells were sorted directly into 350 mL RLT plus buffer (Qiagen) and RNA extracted using the RNeasy® Plus Micro Kit (Qiagen #74034). RNA sequencing was performed by the Yale Center for Genome Analysis using the Illumina HiSeq2500 system, with paired-end 2 x 76 bp read length. Libraries were prepared after poly-A selection