The strong interactions between the probes and target RNA enables washing in high stringency
conditions (3M guanidine thiocyanate at 45°C), thus reducing the potential for false positives due
to non-specific interactions. Finally, we elute bound RNA-chromatin complexes and sequence the
purified genomic DNA.
A. Wash beads
Preheat Wash Buffers at 45°C
Magnetically separate and remove supernatant. (Wash in 1.5mL tube)
Wash 6x 45°C with 500uL 3M GuSCN wash buffer for 3-4 minutes
Transfer to new tube before removing final wash.
B. Elution and Reverse Crosslinking
Remove and discard final wash.
Add 325uL NLS Elution Buffer, 25uL 5M NaCl, 50uL ProK to each sample.
Incubate overnight at 60°C in the thermal cycler.
C. Aliquot Samples
Transfer samples off beads.
Note: Save half (200uL) for pre-library DNA backup.
D. SPRI Cleanup
Add 2.2X volume of SPRI Beads (220ul)
Mix well by pipetting and incubate at RT for 2 minutes
Place on magnet for 4 minutes
Remove supernatant
Add 100ul of 70% EtOH while on magnet
Incubate for 30 seconds and remove EtOH
Remove plate from magnet and allow to dry at RT for 5 minutes
Elute in 33.2ul of H2O, remove from bead.