Illumina NovaSeq 6000 paired end sequencing; ATAC-seq of HSCs in Vitamin A-free diet
ATAC-seq of HSCs in Vitamin A-free diet
3-4-week old C57BL/6J mice were kept in a vitamin A-free diet (0 IE/g vitamin A) or control food (25 IE/g vitamin A) (ssniff Spezialdiäten GmbH) for 16 to 24 weeks. Subsequently, 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 or BV650 or FITC; anti-CD117/c-Kit (2B8)-PE or BV711; anti-Ly6a/Sca-1 (D7)-APCCy7; anti-CD34 (RAM34)-FITC or AF700; anti-CD150 (TC15-12F12.2)-BV605 or PECy5; anti-CD48 (HM48-1)-BV421 or PECy7. 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: 100 m nozzle; 10,000 evt/s.; 70 psi. Each 1500 LT-HSCs (Lin-negative, Sca1-positive, c-Kit-positive, CD150-positive, CD48-negative, CD34-negative) were sorted, respectively, in 3 biological replicates from control and vitamin A deficient animals. Cells were sorted into PBS 1%FCS, spun down and resuspended in 20ul transposition buffer (1ul Tn5, 10ul 2x TD buffer (Illumina), 0.01% digitonin, 0.3x PBS). Transposase reaction was done for 30min at 37°C before DNA was isolated by Qiagen MinElute Clean up kit. Libraries were amplified at 15 cycles and DNA was isolated using AMPure XP beads (Beckman Coultier).