NEXTflex™ Rapid Directional qRNA-Seq™ Kit
For each reaction combine the following in a nuclease-free microcentrifuge tube or plate:14 µL RNA (in Nuclease-free Water or Elution Buffer); 5 µL NEXTflex™ RNA Fragmentation buffer.
Mix thoroughly by pipetting.
Heat for 10 minutes at 95°C, immediately place on ice.
For each reaction, add 1 µL NEXTflex™ First Strand Synthesis Primer to the fragmented RNA (from Section "RNA Fragmentation").
Heat at 65°C for 5 minutes, immediately place on ice.
For each reaction, combine the following in a nuclease-free microcentrifuge tube or plate: 20 µL Fragmented RNA + NEXTflex™ First Strand Synthesis Primer; 4 µL NEXTflex™ Directional First Strand Synthesis Buffer Mix; 1 µL NEXTflex™ Rapid Reverse Transcriptase.
Mix thoroughly by pipetting.
Incubate at 25°C for 10min.
Incubate at 50°C for 50min.
Incubate at 70°C for 15min.
For each reaction, combine the following in a nuclease-free microcentrifuge tube or plate: 25 µL First Strand Synthesis product; 25 µL NEXTflex™ Directional Second Strand Synthesis Mix (contains dUTP).
Mix thoroughly by pipetting.
Incubate for 60 minutes at 16°C.
Add 90 µL of well mixed AMPure XP Beads to each well containing sample.
Mix thoroughly by pipetting.
Incubate the plate for 5 minutes at room temperature.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Remove and discard all of the supernatant from the plate taking care not to disturb thebeads.
With plate on stand, add 200 µL of freshly prepared 80% ethanol to each well without disturbing the beads and incubate the plate for at least 30 seconds at room temperature.
Carefully, remove and discard the supernatant.
Repeat step 18, for a total of two ethanol washes.
Ensure the ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 2 minutes.
Resuspend dried beads in 17 µL of Resuspension Buffer.
Mix thoroughly by pipetting.
Ensure that the beads are completely rehydrated and re-suspended.
Incubate resuspended beads at room temperature for 2 minutes.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Transfer 16 µL of the clear supernatant to a fresh well for the next step.
The procedure may be stopped at this point and the reactions stored at -20°C.
For each sample, combine the following reagents on ice in a nuclease-free 96 well PCR Plate: 16 µL Second Strand Synthesis product (from Step "Bead Cleanup"); 4.5 µL NEXTflex™ Adenylation Mix.
Mix thoroughly by pipetting.
Incubate at 37°C for 30min.
Incubate at 70°C for 5min.
For each sample, combine the following reagents on ice in a nuclease-free 96 well PCR Plate: 20.5 µL 3' Adenylated DNA (from Section "Adenylation"); 27.5 µL NEXTflex™ Ligation Mix; 2.0 µL NEXTflex™ Molecular Index Adapters (1 µM).
Mix thoroughly by pipetting.
Incubate on a thermocycler for 10 minutes at 30°C.
Add 40 µL of well mixed AMPure XP Beads to each well containing sample.
Mix thoroughly by pipetting.
Incubate the plate for 5 minutes at room temperature.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Remove and discard all of the supernatant from the plate taking care not to disturb the beads.
With plate on stand, add 200 µL of freshly prepared 80% ethanol to each well without disturbing the beads and incubate the plate for at least 30 seconds at room temperature.
Carefully, remove and discard the supernatant.
Repeat step 37, for a total of two ethanol washes.
Ensure the ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 2 minutes.
Resuspend dried beads in 51 µL of Resuspension Buffer.
Mix thoroughly by pipetting.
Ensure that the beads are completely rehydrated and re-suspended.
Incubate resuspended beads at room temperature for 2 minutes.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Transfer 50 µL of the clear supernatant to a fresh well.
Add 40 µL of well mixed AMPure XP Beads to each well containing sample.
Mix thoroughly by pipetting.
Incubate the plate for 5 minutes at room temperature.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Remove and discard all of the supernatant from the plate taking care not to disturb the beads.
With plate on stand, add 200 µL of freshly prepared 80% ethanol to each well without disturbing the beads and incubate the plate for at least 30 seconds at room temperature.
Carefully, remove and discard the supernatant.
Repeat step 48, for a total of two ethanol washes.
Ensure the ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 2 minutes.
Resuspend dried beads in 34 µL of Resuspension Buffer.
Mix thoroughly by pipetting.Ensure that the beads are completely rehydrated and re-suspended.
Incubate resuspended beads at room temperature for 2 minutes.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Transfer 33 µL of the clear supernatant to a fresh well for the next step.
The procedure may be stopped at this point and the reactions stored at -20°C.
For each sample (from Section "Bead Cleanup 2"), add 1 µL NEXTflex™ Uracil DNA Glycosylase and mix.
Incubate at 37°C for 30 minutes.
Incubate at 98°C for 2 minutes, then transfer to ice.
For each sample, combine the following reagents on ice in a nuclease-free 96 well PCR Plate: 34 µL Glycosylase-treated Adapter Ligated DNA; 12 µL NEXTflex™ PCR Master Mix; 2 µL NEXTflex™ qRNA-Seq™ Universal Forward Primer; 2µL NEXTflex™ qRNA-Seq™ Barcoded Primer.
Mix thoroughly by pipetting.
PCR cycles: Add 40 µL of well mixed AMPure XP Beads to each well containing sample.
Mix thoroughly by pipetting.
Incubate the plate for 5 minutes at room temperature.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Remove and discard all of the supernatant from the plate taking care not to disturb the beads.
With plate on stand, add 200 µL of freshly prepared 80% ethanol to each well without disturbing the beads and incubate the plate for at least 30 seconds at room temperature.
Carefully, remove and discard the supernatant.
Repeat step 66, for a total of two ethanol washes.
Ensure the ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 2minutes.
Resuspend dried beads in 51 µL of Resuspension Buffer.
Mix thoroughly by pipetting.
Ensure that the beads are completely rehydrated and re-suspended.
Incubate resuspended beads at room temperature for 2 minutes.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Transfer 50 µL of the clear supernatant to a fresh well.
Add 40 µL of well mixed AMPure XP Beads to each well containing sample.
Mix thoroughly by pipetting.
Incubate the plate for 5 minutes at room temperature.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Remove and discard all of the supernatant from the plate taking care not to disturb the beads.
With plate on stand, add 200 µL of freshly prepared 80% ethanol to each well without disturbing the beads and incubate the plate for at least 30 seconds at room temperature.
Carefully, remove and discard the supernatant.
Repeat step 77, for a total of two ethanol washes.
Ensure the ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 2minutes.
Resuspend dried beads in 17 µL of Resuspension Buffer.
Mix thoroughly by pipetting.
Ensure that the beads are completely rehydrated and re-suspended.
Incubate resuspended beads at room temperature for 2 minutes.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Transfer 16 µL of the clear supernatant to a fresh well.
Proceed to cluster generation or store at -20°C.
