PENN SCAP-T Protocols

Library prep protocol

Version 2

University of Pennsylvania

Edited from Truseq stranded-removed frag step

Truseq Stranded Library Prep

Step 1: Fragment RNA

Step 2: First Strand

Step 3: Second Strand/End Repair

Step 4: Adenylate 3’ Ends

Step 5: Ligate Adapters

Step 6: Enrich DNA Fragments

 

 

 

Fragment RNA

  • Use 400ng RNA in 5μl water
  • Add 13μl elute, prime, fragmentation mix

First Strand

  • Transfer 17μl of soln to new tube
  • Briefly spin first strand master mix
  • Add 50μl Superscript III to first strand Act D mix (1SSIII:9FS)
  • Mix gently and spin
  • Add 8μl of mix to each tube, pipette 6x to mix, spin
  • Incubate 25° 10 mins
  • Incubate 42° 15 mins
  • Incubate 70° 15 mins
  • Place on ice

Second Strand/End Repair

  • Briefly spin second strand master mix
  • Add 5μl resuspension buffer
  • Add 20μl of second strand mix to each tube, pipette 6x to mix
  • Incubate 16 degrees 1 hr
  • Let come to RT
  • Cleanup
    • Vortex beads
    • Add 90μl beads to each tube
    • Pipette up and down 10x
    • Incubate at RT 15 mins
    • Place on magnetic stand, wait 5 mins
    • Remove and discard 135ul supernatant
    • Add 200μl freshly prepared 80% EtOH (without disturbing beads)
    • Incubate at RT 30 seconds
    • Remove supernatant (without disturbing beads)
    • Repeat 80% EtOH wash
    • Let stand at RT 15 mins to dry, then remove from magnetic stand
    • Briefly spin the RT resuspension buffer
    • Add 17.5μl resuspension buffer, pipette 10x to mix
    • Incubate at RT 2mins
    • Place on magnetic stand 5 mins
    • Transfer 15μl supernatant to new tubes

Adenylate 3’ Ends

  • Add 12.5μl A-tailing mix to each tube
  • Add 2.5μl resuspension buffer to each tube
  • Incubate 37 degrees 30 mins
  • Incubate 70 degrees for 5 mins
  • Place on ice

Ligate Adapters

  • Add 2.5μl Ligation mix to each tube
  • Add 2.5μl RNA adapter index to each tube
  • Add 2.5μl resuspension buffer to each tube
  • Set pipette to 40μl and mix up and down 10x
  • Incubate 30 degrees 10 minutes
  • Add 5μl stop ligation buffer and pipette up and down 10x
  • Cleanup
    • First wash
      • Vortex beads
      • Add 42μl beads to each tube
      • Pipette up and down 10x
      • Incubate at RT 15 mins
      • Place on magnetic stand, wait 5 mins
      • Remove and discard 79.5μl supernatant
      • Add 200μl freshly prepared 80% EtOH (without disturbing beads)
      • Incubate at RT 30 seconds
      • Remove supernatant (without disturbing beads)
      • Repeat 80% EtOH wash
      • Let stand at RT 15 mins to dry, then remove from magnetic stand
      • Briefly spin the RT resuspension buffer
      • Add 52.5μl resuspension buffer, pipette 10x to mix
      • Incubate at RT 2mins
      • Place on magnetic stand 5 mins
      • Transfer 50μl supernatant to new tubes
    • Second wash
      • Vortex beads
      • Add 50μl beads to each tube
      • Pipette up and down 10x
      • Incubate at RT 15 mins
      • Place on magnetic stand, wait 5 mins
      • Remove and discard 95μl supernatant
      • Add 200μl freshly prepared 80% EtOH (without disturbing beads)
      • Incubate at RT 30 seconds
      • Remove supernatant (without disturbing beads)
      • Repeat 80% EtOH wash
      • Let stand at RT 15 mins to dry, then remove from magnetic stand
      • Briefly spin the RT resuspension buffer
      • Add 22.5μl resuspension buffer, pipette 10x to mix
      • Incubate at RT 2mins
      • Place on magnetic stand 5 mins
      • Transfer 20μl supernatant to pcr tubes

Enrich DNA Fragments

  • Thaw to RT and briefly centrifuge PCR mastermix and PCR primer cocktail
  • Add 5μl PCR primer cocktail to each tube
  • Add 25μl PCR Master mix to each tube, pipette 10x to mix
  • Run PCR program
    • 98° for 30s.
    • 13 cycles
      • 98° for 10 s
      • 60° for 30s
      • 72° for 30s
    • 72 degrees for 5 mins
    • Hold at 4° 
  • Cleanup 
    • Vortex beads 
    • Add 50μl beads to each tube 
    • Pipette up and down 10x 
    • Incubate at RT 15 mins 
    • Place on magnetic stand, wait 5 mins 
    • Remove and discard 95μl supernatant 
    • Add 200μl freshly prepared 80% EtOH (without disturbing beads) 
    • Incubate at RT 30 seconds 
    • Remove supernatant (without disturbing beads) 
    • Repeat 80% EtOH wash 
    • Let stand at RT 15 mins to dry, then remove from magnetic stand 
    • Briefly spin the RT resuspension buffer 
    • Add 52.5μl resuspension buffer, pipette 10x to mix 
    • Incubate at RT 2mins 
    • Place on magnetic stand 5 mins 
    • Transfer 50μl supernatant to new tubes 
  • Cleanup 
    • Vortex beads 
    • Add 50μl beads to each tube 
    • Pipette up and down 10x 
    • Incubate at RT 15 mins 
    • Place on magnetic stand, wait 5 mins 
    • Remove and discard 95μl supernatant 
    • Add 200μl freshly prepared 80% EtOH (without disturbing beads) 
    • Incubate at RT 30 seconds 
    • Remove supernatant (without disturbing beads) 
    • Repeat 80% EtOH wash 
    • Let stand at RT 15 mins to dry, then remove from magnetic stand 
    • Briefly spin the RT resuspension buffer 
    • Add 22.5μl resuspension buffer, pipette 10x to mix
    • Incubate at RT 2mins 
    • Place on magnetic stand 5 mins 
    • Transfer 20μl supernatant to new tubes 

Expect 260bp libraries 

 

 

 

 

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