GSM4475475: 538_ML1_nes-msc_early_ch+; Mus musculus; RNA-Seq
bones were dissected in small pieces of 2-3 mm diameter and incubated 30 min at 37 degrees in a HBSS digestion solution containing 2% FBS, DNase I (10 μg/ml, Sigma), Collagenase (1 mg/ml, Sigma), Dispase II (5 mg/ml, Sigma) and Heparin (20 U/ml, Sigma). Bone pieces were next crashed with mortar and pestle in the same digestion solution and incubated at 37 degrees for 30 min. Cell suspension was then homogenized with a micropipette and filtered with a 100 micron cell strainer. Human and Murine hematopoietic cells were eliminated using magnetic beads isolation kits, and CD31+ labelled endothelial cells were isolated via FACS in RNA extraction buffer. Total RNA from sorted cell samples was isolated using the RNeasy Mini Kit (Qiagen, Crawley, UK). The quality and concentration of total RNA were determined on Agilent 2100 Bioanalyzer using the Eukaryote Total RNA Pico Assay. 2 ng of Total RNA in 5 μl volume was used to generate cDNA synthesis with Nugen Ovation® RNA‑Seq System V2 kit (part No. 7102). The resulting SPIA-cDNA were normalized to 100 ng in 15 μl based on Qubit DNA HS assay. Fragmentation was done using 8 microTUBE-15 AFA Beads Strip V2 (PN 520159) on Covaris E-series at setting 20%DF, 18W, 200 burst, 20C tempt, 120s treatment time and no Intensifier. Only 10-30 ng in 10 μl of fragmented cDNA was used starting from the End Repair step of the Ovation® Ultralow Library Systems V2 (part No. 0344NB) protocol, with 10 cycles PCR. The RNAseq libraries were quality checked on Qubit DNA HS assay, Bioanalyser and Illumina Ecoreal QPCR followed by Illumina PE51 sequencing on Hiseq 2500 V3 reagents.