GSM6630485: WT S2; Mus musculus; RNA-Seq
Spine, hips, femur, tibia, sternum and humerus were harvested. For each mice, bones were pooled and bone marrow (BM) extracted by crushing the bones in cold PBS followed by filtration with 40µm cell strainer.  After erythrocyte lysis, cells were stained with antibody mixes and FACS-sorted using BD Aria III cell sorter. Total RNA was isolated using the Arcturus Picopure RNA isolation kit (Arcturus – Thermo Fisher) from small numbers of FACS-sorted cells (27000 – 75000). RNA was amplified with the NEBNext Single cell/Low input RNA libray kit v. 3.0 (NEB#E6420) following the protocol to the fragmentation step. cDNA was fragmented to target size of 150 base pairs with Covaris M220. Libraries were prepared with TruSeq RNA library preparation kit v. 2.0 (RS#122-2001, Illumina) starting the protocol from “perform end repair” to final library. Libraries were sequenced with the NextSeq550 instrument (Illumina) at the GSCT (UiT – The Arctic University of Norway). 3-4 biological replicates were used per experimental group. The quantity of the Illumina libraries was determined using dsDNA - Qubit High Sensitivity and fragment size was determined by Agilent Technologies 2100 Bioanalyzer using the Agilent DNA 1000 chip.