Matt:LabNotes/2016-8-21

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Revision as of 05:49, 23 August 2016 by >Mzcai (→‎V6 Padlock Probe Capture)
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SplintR Ligase Test

Reference

Experimental Outline

  1. Remove DNA from RNA (UHRR 740000-41)
    • Shouldn't be completely necessary
  2. Biotinylate RNA
  3. Attach RNA to magnetic streptavidin beads
  4. V6 Padlock Probe capture to RNA
  5. Quantify captured products by qPCR
    • Also adds Illumina sequencing adapters

Sample Groups

  • Use Universal Human Reference RNA (UHRR 740000-41)
  • 3 RNA template concentrations for each (30ng, 150ng and 820ng)
    • For DNA template positive control only do 1 sample using 300ng gDNA
  • Sample 1: gDNA template captured by V6 padlock probes with Ampligase at 55C
    • Standard padlock probe capture except keep at 55C whole time
  • Sample 2: RNA template captured by V6 padlock probes with SplintR at 25C
    • Should be ligated but non-specific because low temp
  • Sample 3: biotinylated and streptavidin bead linked RNA captured by V6 padlock probes with SplintR at 25C
    • No washing or pull down
    • Should be same as Sample 2 unless RNA is lost during biotin or streptavidin bead steps
  • Sample 4: RNA template captured by V6 with Ampligase at 55C
    • Negative control to see difference between SplintR and Ampligase
  • Sample 5: No template with V6 and SplintR at 25C
    • NTC checks for ssDNA ligase activity
  • Sample 6: biotinylated and strept bead linked RNA captured by V4 padlock probes at 55C followed by wash and then SplintR & gDNA at 25C
    • V4 padlock probes should be targeting wrong strand and completely washed away before
  • Sample 7: biotinylated and strept bead linked RNA captured by V6 at 55C followed by wash and then SplintR at 25C
  • Summary:
    • 3 samples with biotinylated RNA
    • 2 samples where RNA is pulled down by magnetic beads and washed to remove non-specific binding

Experiment

Remove DNA from RNA

  • Skipped this step

Biotinylate RNA

  • Use Pierce RNA 3' End Biotinylation Kit (>75% ligation efficiency)
  • 3ul of 1ug/ul UHRR
  1. Thaw all kit components except 30% PEG and DMSO on ice
    • Thaw DMSO at RT and 30% PEG at 37C for 5-10min
  2. Adjust heating block to 85C
  3. Transfer 3ul RNA and 1ul DMSO to 1.5ml tube and heat for 5min at 85C
  4. Place RNA immediately on ice
  5. Prepare labeling reaction mix by adding in order listed
    • RNA volume was < 4ul due to evaporation
    • Total volume < 30ul and probably had some losses when moving to 0.2ml tube because needed thermalcycler for 16C
    • Use new pipette tip to mix reaction mix after PEG addition
Component Volume
nf H2O 4
10X RNA Ligase Buffer 3
Rnase Inhibitor 1
RNA (w/ 25% DMSO) 4
Biotinylated Cytidine (Bis)phosphate 1
T4 RNA Ligase 2
30% PEG 15
Total 30
  1. Incubate at 16C for overnight (~15 hrs)
  2. Add 70ul of nf-H2O
  3. Add 100ul chloroform:isoamyl alcohol to extract RNA ligase
    • 96ul chloroform + 4ul isoamyl alcohol
    • Vortex briefly and then centrifuge 2-3 min at high speed to separate phases
  4. Carefully remove the top (aqueous) phase and transfer to a new tube
  5. Add 10ul 5M NaCl, 1ul glycogen, and 300ul ice-cold 100% EtOH
  6. Precipitate for 1hr at -20C
  7. Centrifuge for 15min at > 13,000 x g at 4C
  8. Remove supernatant
  9. Wash pellet with 300ul ice-cold 70% EtOH and remove EtOH
  10. Air-dry for ~5min
  11. Resuspend pellet in 200ul nf-H2O

Attach RNA to Streptavidin Beads

  • MyOne C1 protocol
  • Make Solutions A & B and Binding&Wash Buffer
    • Solution A (DEPC-treated 0.1 M NaOH, DEPC-treated 0.05 M NaCl): 2.5ml 1M NaOH + 0.25ml 5M NaCl + 22.25ml nf-H2O
      • NaOH: 39.997g/mol -> 1M NaOH = 0.1g NaOH dissolved in 2.5mL nf H2O
    • Solution B (DEPC-treated 0.1 M NaCl): 0.5ml 5M NaCl + 24.5ml nf-H2O
    • 2X B&W Buffer (10 mM Tris-HCl 1 mM EDTA 2M NaCl): 10ml 5M NaCl + 50ul 0.5M EDTA + 62.5ul 4M Tris-HCl + 14.8875ml H2O
  1. Resuspend beads by vortexing 30sec
  2. Transfer 100ul of beads (10ug/ul) to new tube
  3. Add 1ml 1X B&W Buffer and mix
  4. Pull down by magnet 2min and remove supernatant
  5. Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant
  6. Wash 2x with 100ul Solution A for 2 min each
  7. Wash 2x with 100ul Solution B
  8. Resuspend the beads in 200ul 2X B&W Buffer
  9. Add 200ul biotinylated RNA
  10. Incubate 15min at RT gently rotating
  11. Pull down with magnet for 3min
  12. Wash 3x with 1X B&W Buffer
  13. Resuspend in 60ul nf H2O

V6 Padlock Probe Capture

  • Dilute 2ul of 1ug/ul UHRR into 40ul total(50ng/ul final conc)
  • V4 padlock probes: 234nM (10.7ng/ul) from Batch 10
  • V6 padlock probes: 854nM
Sample#-RNA V4 Probes (234nM) V6 Probes (854nM) gDNA (80.3ng/ul) RNA (50ng/ul) b-RNA (~50ng/ul) 10X Ampligase Buffer 10X SplintR Buffer Ampligase SplintR H2O Total Overnight Temp
1 0 1 3.8 0 0 3 0 0 0 22.2 30 55
2-30 0 1 0 0.6 0 0 3 0 2 23.4 30 25
2-150 0 1 0 3 0 0 3 0 2 21 30 25
2-820 0 1 0 16.4 0 0 3 0 2 7.6 30 25
3-30 0 1 0 0 0.6 0 3 0 2 23.4 30 25
3-150 0 1 0 0 3 0 3 0 2 21 30 25
3-820 0 1 0 0 16.4 0 3 0 2 7.6 30 25
4-30 0 1 0 0.6 0 3 0 0.5 0 24.9 30 55
4-150 0 1 0 3 0 3 0 0.5 0 22.5 30 55
4-820 0 1 0 16.4 0 3 0 0.5 0 9.1 30 55
5 0 1 0 0 0 0 3 0 2 24 30 25
6-30 2.5 0 0 0 0.6 0 3 0 0 23.9 30 55
6-150 2.5 0 0 0 3 0 3 0 0 21.5 30 55
6-820 2.5 0 0 0 16.4 0 3 0 0 8.1 30 55
7-30 0 1 0 0 0.6 0 3 0 0 25.4 30 55
7-150 0 1 0 0 3 0 3 0 0 23 30 55
7-820 0 1 0 0 16.4 0 3 0 0 9.6 30 55
  • Added 50ul mineral oil on top
  • Magnetic streptavidin beads stick to sides of PCR tube at and above the oil line
  • Overnight ~15 hrs
  • Sample 1: Add 3ul Ampligase mix and incubate at 55C for
    • 1ul Ampligase + 1ul Buffer + 8ul H2O
  • Samples 6 and 7:
    1. Transfer aqueous phase to new PCR tubes
    2. Pull down beads (should have annealed padlock probes attached
    3. Remove supernatant and wash beads twice with 1X SplintR Buffer
    4. Resuspend in 30ul 1X SplintR Buffer with SplintR Ligase (and 300ng gDNA for Samples 6)
      • Samples 6: 2ul SplintR + 3.8ul gDNA + 3ul 10X Buffer + 21.2ul H2O
      • Samples 7: 2ul SplintR + 3ul 10X Buffer + 25ul H2O
  • Other Samples: 94C for 2min and then hold at 4C in fridge
  • Enzyme digest template
    • Exo I/III for DNA
    • RNaseH and Riboshredder for RNA

qPCR