Pooled male Dalotia coriaria for SPRITE chromatin assembly library 16
Beetles were macerated with a glass dounce in 8 ml of 2 mM disuccinimidyl glutarate cross-linking solution at room temperature and rocked gently for 45 min. The cell suspension was pelleted by centrifugation for 8 min at 2500 xg at room temperature, rinsed in PBS and re-pelleted. A 3% paraformaldehyde solution in PBS was added and rocked gently at room temperature for exactly 10 min followed by the addition of 2.5 M glycine solution at room temperature for 5 min to quench the crosslinking reaction. Cells were pelleted by centrifugation at 4 C for 4 min at 2500 x g. The pellet was washed in cold 1x PBS and 0.5% BSA two times, aliquoted, flash frozen in liquid nitrogen and stored at -80 C. Cells pellets were thawed on ice and then lysed using buffers A, B and C in the SPRITE protocol with buffer exchanges following centrifugation at 2500 xg for 8 min. The lysed cells were sonicated at 4 C for 1 min (0.7s on, 3.3s off) with a chilled Branson needle tip sonicator. DNA fragmentation of lysate was performed with the addition of 3 l of Turbo DNase to 5 l of lysate, 2 l of 10X SPRITE DNase Buffer, and 5 l of water at 37C for 17 min to obtain a fragment size distribution between 50 to 1000 bp. The cross-links were then reversed and the remainder of the protocol was followed as previously described. The distribution of cluster sizes and ligation efficiency was checked with a MiSeq run in house prior to shipping the twenty paired-end libraries for sequencing on the HiSeqX by Fulgent Genetics.