Matt:LabNotes/2016-8-21: Difference between revisions
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#Wash pellet with 300ul ice-cold 70% EtOH and remove EtOH | #Wash pellet with 300ul ice-cold 70% EtOH and remove EtOH | ||
#Air-dry for ~5min | #Air-dry for ~5min | ||
#Resuspend pellet in | #Resuspend pellet in 200ul nf-H2O | ||
===Attach RNA to Streptavidin Beads=== | ===Attach RNA to Streptavidin Beads=== |
Revision as of 21:09, 22 August 2016
SplintR Ligase Test
Reference
- V4 and V7 Capture Experiment
- V4 Capture Analysis
- Purify UHRR of DNA
- V6 & V8 Probe Production
- Linking biotin to streptavidin
- NEED to add extra wash steps to remove RNases!
Experimental Outline
- Remove DNA from RNA (UHRR 740000-41)
- Shouldn't be completely necessary
- Biotinylate RNA
- Attach RNA to magnetic streptavidin beads
- V6 Padlock Probe capture to RNA
- 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 95C->55C
- Standard padlock probe capture
- Sample 2: RNA template captured by V6 padlock probes with SplintR at 37C
- 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 37C
- 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 37C
- Negative control to see difference between SplintR and Ampligase
- Sample 5: No template with V6 and SplintR at 37C
- 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 37C
- 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 37C
- 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
- Thaw all kit components except 30% PEG and DMSO on ice
- Thaw DMSO at RT and 30% PEG at 37C for 5-10min
- Adjust heating block to 85C
- Transfer 3ul RNA and 1ul DMSO to 1.5ml tube and heat for 5min at 85C
- Place RNA immediately on ice
- 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 |
- Incubate at 16C for overnight (~15 hrs)
- Add 70ul of nf-H2O
- 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
- Carefully remove the top (aqueous) phase and transfer to a new tube
- Add 10ul 5M NaCl, 1ul glycogen, and 300ul ice-cold 100% EtOH
- Precipitate for 1hr at -20C
- Centrifuge for 15min at > 13,000 x g at 4C
- Remove supernatant
- Wash pellet with 300ul ice-cold 70% EtOH and remove EtOH
- Air-dry for ~5min
- 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
- 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
- Resuspend beads by vortexing 30sec
- Transfer 100ul of beads (10ug/ul) to new tube
- Add 1ml 1X B&W Buffer and mix
- Pull down by magnet 2min and remove supernatant
- Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant
- Wash 2x with 100ul Solution A for 2 min each
- Wash 2x with 100ul Solution B
- Resuspend the beads in 200ul 2X B&W Buffer
- Add 200ul biotinylated RNA
- Incubate 15min at RT gently rotating
- Pull down with magnet for 3min
- Wash 3x with 1X B&W Buffer
- Resuspend in 60ul nf H2O
V6 Padlock Probe Capture
- SplintR at 37C or 25C? 4hrs or overnight?