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 steps 19 - 20 , 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 steps 43 - 44 , 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 steps 59 - 60 , 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 steps 80 - 81 , 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 steps 96 - 97 , 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 .
