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.