Illumina NovaSeq 6000 paired end sequencing; ATAC-seq of LT-HSCs and MPPs
ATAC-seq of LT-HSCs and MPPs
Six month-old C57BL6 mice were sacrificed and 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 2000 LT-HSCs (Lin-negative, Sca1-positive, c-Kit-positive, CD150-positive, CD48-negative, CD34-negative) and 10.000 MPP3/4 (Lin-negative, Sca1-positive, c-Kit-positive, CD150-negative, CD48-positive) were sorted, respectively, in 3 biological replicates. 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 37Â°C before DNA was isolated by Qiagen MinElute Clean up kit. Libraries were amplified at 14 cycles for LT-HSCs and 11 cycles for MPP3/4 and DNA was isolated using AMPure XP beads (Beckman Coultier).