GSM5206721: LS05 rep2; Caenorhabditis elegans; Hi-C
Frozen embryos were then resuspended in and crosslinked with 2% formaldehyde in M9 for another 30 mins. The embryos were spun down at 6000g for 30 sec and washed once with 100mM Tris Cl pH 7.5 and twice with M9. The pellet was resuspended in Nuclei Buffer A (15 mM Tris–HCl pH 7.5, 2 mM MgCl2,0.34 M Sucrose,0.15 mM Spermine, 0.5 mM Spermidine, 1 mM DTT,0.5 mM PMSF (1X Calbiochem Protease Inhibitor cocktail I), 0.25% NP-40, 0.1% Triton X-100), centrifuged at 1000g for 5 minutes at 4C then resuspended in 1.5 mL Nuclei Buffer A. The embryos were then dounced 10X with a loose pestle and 10X with a tight pestle. The nuclei were separated from the cellular debris through spinning down the dounced material at 200G, then collecting the supernatant containing the nuclei into a separate tube. The pellet was resuspended in 1.5mL and the douncing process was repeated four times. Each individual supernatant containing nuclei was checked for quality by DAPI staining and those without debris were pooled and spun down at 1000G for 10 mins at 4C. Approximately 20 ul nuclei pellet were used to proceed to Arima Hi-C per the manufacturer's instructions.  Library preparation was performed using the KAPA Hyper Prep Kit using the protocol provided by Arima.