RNA-Seq of B cells from mus musculus control blood 2
Mouse HSCs (Sca1+cKit+CD48-CD150+) and mouse blood cells (T cells: Gr1-CD11b-B220-CD4+CD8+; B cells: Gr1-CD11b-B220+CD4-CD8-; Gr1-CD11b+ cells and Gr1+CD11b- cells) were isolated by FACS from bone marrow and peripheral blood respectively. Total RNA from these cells was isolated with RNeasy microKit (Qiagen). For the construction of the non-directional library, messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, the second strand cDNA synthesis was carried out with dTTP followed by end repair, A-tailing, adapter ligation, size selection, amplification and purification.