Illumina NovaSeq 6000 paired end sequencing; ChIPseq for H3K4me3 and H3K27me3 in LT-HSCs and MPPs
ChIPseq for H3K4me3 and H3K27me3 in 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. Prior to sorting stained cells were cross-linked for 10min at room temperature with 1% formaldehyde and subsequently washed in ice-cold PBS. DAPI was added prior to FACS sort and 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 4.000 LT-HSCs (DAPI-negative, Lin-negative, Sca1-positive, c-Kit-positive, CD150-positive, CD48-negative, CD34-negative) and 4.000 MPP3/4 cells (DAPI-negative, Lin-negative, Sca1-positive, c-Kit-positive, CD150-negative, CD48-positive) were sorted, respectively, in 3 biological replicates. Cells were sorted into 200 ul RIPA buffer. Nuclei were broken by mild sonication (Diagenode Bioruptor, 3 cycles, low power, 30 sec ON/OFF) and chromatin was incubated with antibody-coupled protein G dynabeads (Thermofisher) over night at 4째C. Chromatin was then fragmented by incubation with 1ul Tn5 enzyme in 20ul 1x transposition buffer (Illumina) for 40min at 37째C. Stringent washing steps with low salt (once) and high salt RIPA (four times) as well as TE buffer (twice) were performed to remove all fragmented chromatin that is not associated to the antibody-bead-complex (H3K27me3 Cell Signaling C36B11, H3K4me3 Millipore 07-473). Reverse crosslinking was performed with proteinase K at 60째C over night before DNA was isolated using phenol-chloroform extraction and subsequent ethanol precipitation over night at -80째C. Sequencing libraries were amplified using unique dual barcode primers (Illumina) and 14 PCR cycles. Libraries were cleaned up using AMPure XP beads (Beckman Coultier). Double-sided size selection was performed by removing large fragments that precipitate with 0.2x volume and purifying fragments that precipitate with 1.0x volume ratio. Clean-up was done twice in order to efficiently remove primers and adaptor dimers.