Modified protocol for multiple rounds of PolyA selection
Aliquot and Wash magnetic beads
Wash 2µl MB twice with 50µl BW.
Withdraw and discard supernatant.
Resuspend washed beads in 5µl HYB.
Denature RNA
Prepare total RNA in a volume of 15µl.
Incubate for 1min at 60°C and hold at 25°C.
Hybridize Poly(A) RNA
Add denaturated RNA (15µl) to 5µl washed beads.
Incubate for 20 min at 25°C / 1,250 rpm.
Wash 2X for 5 min at 25°C / 1,250 rpm with BW.
Withdraw and discard supernatant.
Elute and Re-bing Poly(A) RNA
Add 15µl H20.
Incubate for 1 min at 70°C.
Add 5µl HYB to re-bind the polyA+ RNA to the beads.
Incubate for 20 min at 25°C / 1,250 rpm .
Wash 2X for 5 min at 25°C / 1,250 rpm with BW.
Withdraw and discard supernatant.
Elute Poly(A) RNA
Add 25µl H20.
NOTE: When working with input amount <500ng or for CORALL lib prep, only add 12µl of H20.
Incubate for 1 min at 70°C.
Immediately place on magnet for 5 min or until supernatant is clear.
Transfer the clear supernatant into a fresh tube.