Illumina NovaSeq 6000 paired end sequencing; RNA Seq of 7-month old control and Cyp26b1KO LT-HSCs
RNA Seq of 7-month old control and Cyp26b1KO LT-HSCs
Cyp26b1 knockout using the MxCre driver was performed by intraperitoneal injection of 100ug poly I:C /mouse every other day over 10 days (5 injections total). Control mice had floxed Cyp26b1 allels but no MxCre. After a recovery phase of 4 weeks, mice were aged for 6 months and murine bone marrow (BM) cells were isolated from pooled femura, tibiae, hips, ilia, and vertebrae 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 and progenitor 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-CD34 (RAM34)-FITC; 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 III  (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 3000 LT-HSCs (Lin-negative, Sca1-positive, c-Kit-positive, CD150-positive, CD48-negative, CD34-negative) of control and Cyp26b1KO mice were sorted in 2 biological replicates. Sorted cells were collected into ice-cold RNA lysis buffer (ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen)) and stored at -80C until further usage. Total RNA isolation was performed from the indicated populations using ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen) according to the manufacturers instructions. DNAse treatment was performed using RNAse-free DNAse Set (Qiagen). 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, 12 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 manufacturerâ€™s protocol together with unique dual indexing barcodes (NEB, #E6440). Libraries were amplified with 7 PCR-cycles before Ampure bead (Beckman-Coultier) clean-up and subsequent quality control.