Illumina NovaSeq 6000 paired end sequencing; RNA Seq of wildtype and Rarb KO HSCs after 24h of in vitro culture and treatment
RNA Seq of wildtype and Rarb KO HSCs after 24h of in vitro culture and treatment
Murine bone marrow (BM) cells were isolated from pooled femura, tibiae, hips, ilia, and vertebrae of 8week-old WT and RarbKO littermate mice by gentle crushing in PBS using a mortar and pistil. Erythrocyte lysis was performed using ACK Lysing Buffer (Thermo Fisher Scientific). To enrich for lineage negative (Lin-) cells we used the Dynabeads Untouched Mouse CD4 Cells Kit (Invitrogen). Briefly, the ery-lysed BM was stained for 30-45 min with a 1:4 dilution of the Lineage Cocktail provided in the Dynabeads Untouched Mouse CD4 Cells Kit (Invitrogen). Labelled cells were then incubated for 20 min with 400uL/mouse of washed polyclonal sheep anti-rat IgG coated Dynabeads provided in the Kit. Depletion of Lin+ cells was performed using a magnet. To achieve further purification of hematopoietic stem cells we used fluorescence- activated cell sorting (FACS). Therefore, the depleted cell fraction was stained for 30 min to 1h using the following monoclonal antibodies: anti-lineage [anti-CD4 (clone GK1.5), anti-CD8a (53-6.7), anti-CD11b (M1/70), anti- B220 (RA3-6B2), anti-GR1 (RB6-8C5) and anti-TER119 (Ter-119)] all PE-Cy7; anti-CD117/c-Kit (2B8)-PE; anti-Ly6a/Sca-1 (D7)-APCCy7; anti-CD150 (TC15-12F12.2)-BV605; anti-CD48 (HM48-1)-BV421. Monoclonal antibody conjugates were purchased from eBioscience or BioLegend. Sorting was then performed on a FACS Aria II, III or Symphony (Becton Dickinson) at the MPI FACS facility, Freiburg, Germany. The following sort parameters were used: 70 m nozzle; 10,000 evt/s.; 70 psi.  Each 1000 HSCs (Lin-negative, Sca1-positive, c-Kit-positive, CD150-positive, CD48-negative) were sorted per well of a 96well ultra-low attachment plate into 100ul of Complete Stem Cell Medium (StemPro®-34 SFM, LifeTechnologies containing 50 ng/ml SCF, 25 ng/ml TPO, 30 ng/ml Flt3-Ligand (all Preprotech), 100 ml/ml Penicillin/Streptomycin, 2 mM L-Glutamine (both Gibco)) and cultured for 24h with either at-RA (5uM final) (Sigma-Aldrich) or 4-oxo-RA (5uM final) (Santa Cruz)  or the respective amount of DMSO. Cells were harvested and total RNA was purified using ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen) according to the manufacturers instructions. Total RNA was used as starting material for cDNA-libraries preparation using the SMARTseq v4 Ultra Low RNA Kit (Takara) according to the manufacturers indications. Of note, 13 cycles were used for the amplification of cDNA. Sequencing libraries were prepared from 10ng of cDNA libraries as input using NEBNext Ultra II FS DNA library prep kit (#E7805S, NEB) according to manufacturer's protocol together with unique dual indexing barcodes (NEB, #E6440). Libraries were amplified with 8 PCR-cycles before Ampure bead (Beckman-Coultier) clean-up and subsequent quality control.