Revision as of 22:42, 29 August 2016 by >Mzcai
SplintR Ligase Test
Reference
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 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
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
- 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
- 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 21 hrs
- Sample 1: Add 3ul Ampligase mix and incubate at 55C for 21 hrs
- 1ul Ampligase + 1ul Buffer + 8ul H2O
- Samples 6 and 7:
- Transfer aqueous phase to new PCR tubes
- Beads stuck to original tube so lost a lot during transfer
- Pull down beads (should have annealed padlock probes attached)
- Remove supernatant and wash beads twice with 100ul 1X SplintR Buffer
- 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
- Incubate at 25C for 21 hrs
- 94C for 2min and then hold at 4C for 3 hrs (went to lunch)
- Other Samples: 94C for 2min and then hold at 4C in fridge for 21 hrs
- Take out 10ul from each tube and go straight to qPCR
- Enzyme digest template from ~15ul that is left in tubes
- 2ul Exo I/III (1:1) for DNA (Samples 1 & 6)
- 2ul RNaseH and Riboshredder (1:1) for RNA (Samples 2-5 & 7)
- 37C for 1 hr
- First add 1ul 5M NaCl (100nM NaCl or KCl for Riboshredder)
- 94C for 2min
qPCR
Primers
Primer
|
Sequence
|
Index #
|
ISB_CA_AF |
AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG |
|
ISB_CA_AR.T1 |
CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG |
Indx1
|
ISB_CA_AR.T2 |
CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG |
Indx2
|
ISB_CA_AR.T3 |
CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG |
Indx3
|
PCR Test for Non-Enzyme Digested Samples
Components
|
1X Volume
|
18X Volume
|
Captured template |
1 |
0
|
10uM ISB_CA_AF |
0.4 |
7.2
|
10uM ISB_CA_AR.T1 |
0.4 |
7.2
|
2X KAPA SYBG MM |
12.5 |
225
|
H2O |
10.7 |
192.6
|
Total |
25 |
432
|
- Aliquot 24ul from 18X master mix and add 1ul captured template
Program
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
PCR Test for Enzyme Digested Samples
Components
|
1X Volume
|
18X Volume
|
Captured template |
1 |
0
|
10uM ISB_CA_AF |
0.4 |
7.2
|
10uM ISB_CA_AR.T1 |
0.4 |
7.2
|
2X KAPA SYBG MM |
12.5 |
225
|
H2O |
10.7 |
192.6
|
Total |
25 |
432
|
- Aliquot 24ul from 18X master mix and add 1ul captured template
Program
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min