TruSeq RNA kit protocol ( with one - third reaction volumes and some minor changes )
If not performing Poly A select mRNA , proceed directly to step 24 .
Mix 500 ng total RNA and dH2O to a final volume of 16.67 uL .
Vortex RNA purification Beads and add 16.67 uL to RNA sample .
Mix by pipetting up and down until beads are in a homogenous suspension .
Incubate in thermocycler : 65 °C 5 min 4 °C hold .
When thermocycler reaches 4 °C remove sample and place on bench at room temperature for 5 min .
Place sample in magnetic rack for 5 min .
Remove and discard all the supernatant .
Remove sample from rack .
Add 66.7 uL of Bead Washing Buffer and pipet up and down until beads are in a homogenous suspension .
Place the sample back in the magnetic rack for 5 min .
Remove and discard all of the supernatant .
Add 16.67 uL of Elution Buffer and pipet up and down until beads are in a homogenous suspension .
Incubate in thermocycler : 80 °C 2 min 25 °C hold .
Remove sample from thermocycler when it reaches 25 °C and keep at room temp .
Add 16.67 uL of Bead Binding Buffer and pipet up and down until beads are in a homogenous suspension .
Incubate at room temperature for 5 min .
Place sample in magnetic separator for 5 min .
Remove and discard all supernatant .
Remove sample from rack .
Add 66.7 uL of Bead Washing Buffer and pipet up and down until beads are in a homogenous suspension .
Place sample in magnetic separator for 5 min .
Remove and discard all supernatant .
Add 6.5 uL Elute , Prime , Fragment .
Mix and pipet up and down until beads are in a homogenous suspension .
If proceeding directly from step 1 , add total RNA to 6.5 uL of Elute , Prime , and Fragment mix , and bring up the total volume to no more than 10 uL with dH2O .
Incubate in thermocycler : 94 °C 8 min 4 °C hold .
If not performing Poly A select mRNA , proceed directly to step 28 .
Place sample in a magnetic rack for 5 min .
Transfer 5.67 uL of the supernatant to a new 0.2 mL PCR tube .
Add 2.67 uL of First Strand Master Mix / Super Script II mix to sample .
Incubate in thermocycler : 25 °C 10 min 42 °C 50 min 70 °C 15 min 4 °C hold .
Add 8.33 uL of Second Strand Master Mix to sample .
Incubate in thermocycler at 16 °C for 1 hour .
Remove sample from thermocycler and let warm to room temperature .
Add 30 uL of well - mixed AMPure XP beads and mix by pipetting up and down until beads are in a homogenous suspension .
Incubate at room temperature for 15 min .
Place on magnetic rack for 5 min .
Remove and discard 45 uL of the supernatant .
Keep sample in magnetic rack and add 200 uL of 80 % ethanol .
Incubate for 30 seconds .
Remove and discard all supernatant .
Repeat steps 37 and 38 once more for a total of two washes .
Add 22 uL Resuspension Buffer and pipet up and down until beads are in a homogenous suspension .
Incubate at room temperature for 5 min .
Place in magnetic rack for 5 min .
Transfer 20 uL of the supernatant to a new 0.2 mL PCR tube .
Add 13.3 uL of End Repair Mix to sample .
Incubate at 30 °C for 30 min .
Add 53.5 uL of well - mixed AMPure XP Beads and mix by pipetting up and down until beads are in a homogenous suspension .
Incubate at room temperature for 15 min .
Place on magnetic rack for 5 min .
Remove and discard 81.6 uL of the supernatant .
Keep sample in magnetic rack and add 200 uL of 80 % ethanol .
Incubate for 30 seconds .
Remove and discard all supernatant .
Repeat steps 50 and 51 once more for a total of two washes .
Add 7.83 uL Resuspension buffer and mix by pipetting up and down until beads are in a homogenous suspension .
Incubate at room temperature for 5 min .
Place in magnetic rack for 5 min .
Transfer 5.83 uL of the supernatant to a new 0.2 mL PCR tube .
Add 4.17 uL A - Tailing Mix to sample .
Incubate at 37 °C for 30 min .
Add 0.83 uL DNA Ligase .
Mix 0.83 uL Resuspension Buffer 0.83 uL RNA Adapter .
Index and mix by pipet or flicking , and spin down .
Incubate at 30 °C for 10 min .
Add 1.67 uL Stop Ligase Mix .
Add 14 uL well - mixed AMPure XP Beads and mix by pipetting up and down until beads are in a homogenous suspension .
Incubate at room temperature for 15 min .
Place on magnetic rack for at least 5 min .
Remove and discard 23.16 uL of the supernatant .
Keep sample in magnetic rack and add 200 uL of 80 % ethanol .
Incubate for 30 seconds .
Remove and discard all supernatant .
Repeat steps 66 and 67 one more time .
Add 18.67 uL Resuspension Buffer and pipet up and down until beads are in a homogenous suspension .
Incubate at room temperature for 5 min .
Place in magnetic rack for 5 min .
Transfer 16.67 uL of the supernatant to a new 0.2 mL PCR tube .
Add 16.67 uL of well - mixed AMPure XP beads .
Mix by pipetting up and down until the beads are in a homogenous suspension .
Incubate at room temperature for 15 min .
Place on magnetic rack for at least 5 min .
Remove and discard 28.34 uL of the supernatant .
Keep sample in magnetic rack and add 200 uL of 80 % ethanol .
Incubate for 30 seconds .
Remove and discard all supernatant .
Repeat steps 77 and 78 one more time .
Add 9.67 uL Resuspension Buffer and pipet up and down 10 times .
Incubate at room temperature for 5 min .
Place in magnetic rack for 5 min .
Transfer 7.67 uL of the supernatant to a new 0.2 mL PCR tube .
Use 1 uL to determine number of cycles to perform in following PCR amplification .
Mix 6.67 uL Adapter ligated DNA from step 831.67 uL PCR Primer Cocktail 8.33 uL PCR Master Mix .
Amplify with the following PCR procotol 98 °C - 30 seconds 5 - 18 cycles 98 °C - 10 seconds 60 °C - 30 seconds 72 °C - 30 seconds 72 °C - 5 hold at 4 °C .
Add 16.67 uL of well - mixed AMPure XP beads .
Incubate at room temperature for 15 min .
Place on magnetic rack for at least 5 min .
Remove and discard 28.3 uL of the supernatant .
Keep sample in magnetic rack and add 200 uL 80 % ethanol .
Incubate 30 seconds .
Remove and discard all supernatant .
Repeat steps 91 and 92 one more time .
Let the beads dry at room temperature for 2 min .
Add 12 uL Resuspension Buffer and pipet up and down 10 times .
Incubate at room temperature for 2 min .
Place in magnetic rack for 5 min .
Transfer 10 uL of the supernatant to a new 1.5 mL PCR tube .
Use 1 uL for qPCR quantiation ( http : / / ethanomics . wordpress . com / ngs - qpcr - library - quantitation - protocol / ) and run 1 uL on the Bioanalyzer .
