============================================================== Nuclei Isolation for Downstream Genomics Applications ============================================================== Nuclear Isolation ----------------------------------------------- .. time:: 1 - 1.5 hrs depending on the number of samples 1. Prior to starting, make the Lysis Buffer and Wash Buffer and keep them on ice. Be sure to use freshly made Lysis Buffer and Wash Buffer each time – see recipe at the bottom. 2. Obtain 50,000 cells for MEFs by trypsin and count accordingly. - *Optional*: if using a spike-in: Add 1000 cells from HEK293Ts to each MEF tube. 3. Pellet at 500xg for 5 mins at 4C. Aspirate supernatant gently using P1000. 4. Resuspend the cell pellet in 50 µL of Lysis Buffer by pipetting up and down three times. 5. Incubate on ice for 3 min. If lysing multiple samples, make sure that all samples are lysed for the same total amount of time by proceeding to Step 7 after 3 min. 6. Add 1 mL of Wash Buffer to dilute the lysis reagents. Invert the tube five times to mix. 7. Pellet nuclei at 500g for 10 min at 4°C in a fixed-angle microcentrifuge. Orient the tubes in a consistent fashion so that the pellet will end up in the same location. 8. STOPPING POINT: Pelleted nuclei can be flash frozen, fixed or immediately processed for downstream genomic applications like ATAC-seq, ChIP-seq, or Chromatin conformation capture. Recipes ----------------------------------------------- Stock solutions ~~~~~~~~~~~~~~~ Make sure you have enough of the following stock solutions. These are often shared with other recipes. **1M Tris pH 7.5 stock solution**: ======================= ================ ======== ==================== Component Concentration g / L Amount / 10 mL ======================= ================ ======== ==================== Tris-HCl 1 M 157.64 1.57g Elga water to 9 mL NaOH to pH 7.5 ~140 uL of 12 N NaOH Elga water to 10 mL ======================= ================ ======== ==================== **5M NaCl stock solution**: ======================= ================ ======== ==================== Component Concentration g / L Amount / 10 mL ======================= ================ ======== ==================== NaCl 5 M 292.21 2.92g Elga water to 10 mL ======================= ================ ======== ==================== **1M magnesium chloride stock solution**: ======================= ================ ======== ==================== Component Concentration g / L Amount / 10 mL ======================= ================ ======== ==================== MgCl2 1 M 95.21 0.952 g Elga water to 10 mL ======================= ================ ======== ==================== Resuspension Buffer (RSB)- 100mL, scale as needed ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ======================= ==================== ==================== Component Final Concentration Amount / 100 mL ======================= ==================== ==================== 1 M Tris, pH 7.5 10 mM 1 mL 5 M NaCl 10 mM 200 μL 1M MgCl2 3 mM 300 μL Elga water 98.5 mL ======================= ==================== ==================== Cell Lysis Buffer per 50 uL, scale as needed ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ====================================== ================= Component Volume ====================================== ================= Cold RSB (recipe above) 48.5uL 10% (wt/vol) IGEPAL-CA360 0.5uL 10% (wt/vol) Tween-20 0.5uL 1% (wt/vol) digitonin 0.5uL Total volume 50uL ====================================== ================= Wash Buffer per 1000 uL, scale as needed ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ============================== ================= Stock solutions Volume ============================== ================= Cold RSB (recipe above) 990uL 10% (wt/vol) Tween-20 10uL ============================== =================