GSM6935633: GMP_2; Mus musculus; RNA-Seq
Femura, illia and tibiae were isolated from the hind legs and the bones were crushed in a mortar. The bone marrow was re-suspended in PBS/3%FCS and filtered through a 70μm cell strainer (Falcon) to obtain single-cell suspensions.  For cell sorting, bone marrow cells were depleted of lineage positive cells using a Lineage Cell Depletion kit (Miltenyi Biotec, cat-nr.130-090-858) according to manufacturer's instructions, then stained with antibodies of interest (PE anti-mouse EMCN, PE/Cy5 anti-mouse Gr-1, PE/Cy-5 anti-mouse CD11b, PE/Cy5 anti-mouse/human B220, PE/Cy5 anti-mouse CD3e, PE/Cy5 anti-mouse Ter119, Pacific Blue anti-mouse Sca-1, APC-efluor780 anti-mouse c-kit, Alexa Fluor700 anti-mouse CD16/CD32, BV650 anti-mouse CD150, PE/Cy7 CD48) and incubated for 30 min at 4°C. After washing with PBS/3%FCS at 300g for 5 min, 4°C, cells were re-suspended in PBS/3%FCS containing 1:1000 7-Aminoactinomycin D (7-AAD, Lifetechnologies, cat-nr. A1310) for discrimination of dead cells. HSPC populations were sorted on FACS Aria III (BD Biosciences).   RNA was isolated from sorted cells using the RNeasy Plus micro kit (Qiagen). RNA libraries were constructed using the SMARTer stranded total RNA-Seq Kit V2 Pico Input (TaKaRa) according to manufacturer's instructions. Final library library concentrations were measured using Qubit 2.0 Fluorometer in combination with Qubit dsDNA HS Assay Kit (Invitrogen). Size distribution and fragment length were assessed using Agilent Bioanalyzer HS-DNA Chips. Molarity of individual libraries was calculated considering the concentration and average fragment size. Pair-end sequencing of 2.0 pM pooled libraries were performed using an Illumina NextSeq 500 System in combination with NextSeq 500/550 Mid Output v2 kit (150 cycles). PhiX Control (Illumina) was added at 1% to the pool as an internal control before sequencing.