CUT&RUN of erythroblasts upon perturbation of gamma-hemoglobin repressors (part II)
CUT&RUN of erythroblasts upon perturbation of gamma-hemoglobin repressors (part II)
Human CD34+ cells were expanded and differentiated using the previously described 2-phase culture method. Cells were harvested by centrifugation (200 x g for 8 minutes), washed 2 times with PBS, counted with Countess cell counter (Thermo Fischer) and fixed for 5 minutes on a platform shaker with 1% fresh formaldehyde (Pierce 16% Formaldehyde Methanol Free, 28906) diluted in PBS. The fixation was quenched with 1:10 vol of 1.25 M glycine solution for 5 minutes with shaking. All the steps were carried out at room temperature unless specified. Cells were centrifuged at 600 x g for 5 minutes and washed with PBS twice. The cells were resuspended in PBS and transferred into a final volume of 500 L in 1.5 mL Eppendorf tubes pre-coated overnight with 1 % BSA. Aliquots of 2 million-, 500,000-, or 200,000-cells were used for WIZ, CTCF or histone PTM (posttranslational modification) profiling, respectively. Activated BioMag® Plus Concanavalin A magnetic beads (Bangs Laboratories, BP531) were added to the cells (40L per tube) to allow magnetic separation in the following steps and incubated for 10 minutes with gentle rotation. After magnetic separation cells were resuspended in 500L of Permeabilization Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.02 % Digitonin, 2 mM EDTA, 0.1 % NP40, 0.1 % Tween-20, cOmplete EDTA-free Protease Inhibitor Cocktail, 4693132001) and incubated for 7 minutes without shaking. Cells were washed with 1 mL of DIG-wash EDTA Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.02% Digitonin, 2 mM EDTA, cOmplete EDTA-free Protease Inhibitor Cocktail) and incubated in 200L of DIG-wash EDTA Buffer with the primary antibody for 2 hours with gentle rotation. Primary antibodies used were rabbit polyclonal WIZ (Novus Biologicals, NBP1-80586, lot A96963), rabbit polyclonal CTCF (Millipore, 07-729, lot 3059608), rabbit polyclonal H3K9ac (Active Motif, 39917, lot 12517005) or mouse monoclonal H3K9me2 (Millipore, 05-1249, clone CMA307, lot 3385820). Cells were washed twice with 1 mL of DIG-wash EDTA buffer and incubated for 1 hour at 4°C with gentle rotation in 200L of DIG-wash EDTA Buffer with CUTANA pAG-MNase (protein A- protein G-Micrococcal Nuclease fusion, Epicypher, 15-1016). Subsequently, the cells were washed once with 1 mL of DIG-wash EDTA Buffer and once with 1 mL of DIG-wash Buffer without EDTA. For the pAG-MNase digestion, the cells were re-suspended in 100 L of DIG-wash Buffer without EDTA, then CaCl2 was added to a final concentration of 2 mM to activate the MNase. Samples were incubated for 30 minutes at 0 °C in an aluminum block. To stop the digestion, 100L of 2X Stop Buffer (400 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02 % Digitonin, 100 g/mL RNase A, 50g/mL Glycogen, 4 pg/mL Drosophila melanogaster DNA) were added. Samples were incubated for 30 minutes at 37 °C without shaking. Supernatants were collected and treated with SDS (1% final concentration) and 200g/mL Proteinase K for minimum 4 hours at 65°C. DNA was extracted by phenol-chloroform for WIZ and CTCF profiling to keep the small DNA fragments and with the DNA Clean & Concentrator-5 kit (Zymo Research, D4004) for histone PTMs profiling. The NEB adaptor was diluted at 1:15 to avoid excessive adaptor dimer formation. The NEBNext Multiplex Oligos for Illumina SET2 (New England Biolabs, E7500L) was used for the indexing. The PCR reaction was adapted for each target: 8 and 9 cycles and annealing / extension for 10 seconds at 65 °C for WIZ and CTCF, respectively; 9 cycles and annealing / extension for 30 seconds at 65 °C for histone PTMs. Libraries were purified using Agencourt AMPure XP beads (Beckman Coulter, A63881) and the concentrations and profiles were checked on TapeStation with a D1000 Screen Tape.