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=SplintR Ligase Test= ==Reference== *[[Matt:LabNotes/2015-1-21|V4 and V7 Capture Experiment]] *[[Matt:LabNotes/2015-3-19|V4 Capture Analysis]] *[[Matt:LabNotes/2014-9-25#Purify_UHRR_of_DNA|Purify UHRR of DNA]] *[[Matt:LabNotes/2015-1-26|V6 & V8 Probe Production]] *[[Matt:LabNotes/2016-3-31|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 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 <!-- *Use Zymo DNA-Free RNA Kit ====DNase I Digest==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | UHRR (1ug/ul)||3 |- | 10X DNase I Buffer||5 |- | DNase I||2 |- | H2O||39 |- | Total||50 |} *Mix and incubate at 37C for 30min ====Purify==== *Add 100ul RNA Binding Buffer and mix *Add 150ul 100% EtOH and mix *Transfer to Zymo-Spin IC Column in Collection Tube **Centrifuge at 13,000rcf for 1min and discard flow through *Add 400ul RNA Prep Buffer **Centrifuge at 13,000rcf for 1min and discard flow through *Add 800ul RNA Wash Buffer **Centrifuge at 13,000rcf for 30sec and discard flow through *Add 400ul RNA Wash Buffer **Centrifuge at 13,000rcf for 30sec and discard flow through *Centrifuge at 13,000rcf for 2min and discard flow through *Transfer column to 1.5ul tube and add 8ul H2O and let stand for 1min **Centrifuge at 10,000rcf for 30sec *Quantify with Nanodrop or Qubit --> ===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 {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Component | width="51" | Volume |- style="font-size:11pt" valign="bottom" | height="14" | nf H2O | align="right" | 4 |- style="font-size:11pt" valign="bottom" | height="14" | 10X RNA Ligase Buffer | align="right" | 3 |- style="font-size:11pt" valign="bottom" | height="14" | Rnase Inhibitor | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | RNA (w/ 25% DMSO) | align="right" | 4 |- style="font-size:11pt" valign="bottom" | height="14" | Biotinylated Cytidine (Bis)phosphate | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | T4 RNA Ligase | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | 30% PEG | align="right" | 15 |- style="font-size:11pt" valign="bottom" | height="14" | Total | align="right" | 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=== *[https://www.thermofisher.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/protein-isolation-protocol/dynabeads-myone-streptavidin-c1.html|Dynabeads 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: [[Matt:LabNotes/2015-1-21|854nM]] {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" valign="bottom" | width="65" height="16" | Sample#-RNA | width="66" | V4 Probes (234nM) | width="66" | V6 Probes (854nM) | width="66" | gDNA (80.3ng/ul) | width="66" | RNA (50ng/ul) | width="66" | b-RNA (~50ng/ul) | width="66" | 10X Ampligase Buffer | width="66" | 10X SplintR Buffer | width="66" | Ampligase | width="66" | SplintR | width="66" | H2O | width="66" | Total | width="66" | Overnight Temp |- style="font-size:12pt" valign="bottom" | align="right" height="16" | 1 | align="right" | 0 | align="right" | 1 | align="right" | 3.8 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 22.2 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 2-30 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0.6 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 23.4 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 2-150 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 21 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 2-820 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 16.4 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 7.6 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 3-30 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 0.6 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 23.4 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 3-150 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 21 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 3-820 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 16.4 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 7.6 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 4-30 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0.6 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0.5 | align="right" | 0 | align="right" | 24.9 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 4-150 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0.5 | align="right" | 0 | align="right" | 22.5 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 4-820 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 16.4 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0.5 | align="right" | 0 | align="right" | 9.1 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | align="right" height="16" | 5 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 24 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 6-30 | align="right" | 2.5 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 0.6 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 23.9 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 6-150 | align="right" | 2.5 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 21.5 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 6-820 | align="right" | 2.5 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 16.4 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 8.1 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 7-30 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 0.6 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 25.4 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 7-150 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 23 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 7-820 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 16.4 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 9.6 | align="right" | 30 | align="right" | 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) ***37C for 1 hr **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==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Primer''' | align="center" style="background:#f0f0f0;"|'''Sequence''' | align="center" style="background:#f0f0f0;"|'''Index #''' |- | ISB_CA_AF||AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG|| |- | ISB_CA_AR.T1||CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG||Indx1 |- | ISB_CA_AR.T2||CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG||Indx2 |- | ISB_CA_AR.T3||CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG||Indx3 |} <!-- {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Forward Primer''' | align="center" style="background:#f0f0f0;"|'''Reverse Primer''' |- | 1||1||ISB_CA_AF||ISB_CA_AR.T1 |- | 2||2||ISB_CA_AF||ISB_CA_AR.T2 |- | 3||3||ISB_CA_AF||ISB_CA_AR.T3 |- | 4||1||ISB_CA_AF||ISB_CA_AR.T1 |- | 5||2||ISB_CA_AF||ISB_CA_AR.T2 |- | 6||3||ISB_CA_AF||ISB_CA_AR.T3 |}--> ====PCR Test for Non-Enzyme Digested Samples==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''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 [[Media:SplintR_Ligase_Test_qPCR_-_No_Enzyme_Digest.xlsx|Excel sheet with full PCR data]]<br> [[File:SplintR_Ligase_Test_qPCR_-_No_Enzyme_Digest.JPG|450px]] ====PCR Test for Enzyme Digested Samples==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''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 [[Media:SplintR_Ligase_Test_qPCR_-_Enzyme_Digest.xlsx|Excel sheet with full PCR data]]<br> [[File:SplintR_Ligase_Test_qPCR_-_Enzyme_Digest.JPG|450px]] ==Results & Conclusion== *Pos Ctrl (V6 probes capture gDNA with Ampligase) has low amount of ligated padlock probes **High Ct in PCR curve **I guess 300ng gDNA has fewer targets than 30ng RNA **But same low level as Neg Ctrl (Ampligase used on DNA/RNA hybrid) *NTC (V6 captures '''nothing''' with SplintR) has same amount of ligated padlock probes as Experimental samples (V6 captures RNA with SplintR) **suggests SplintR doesn't need RNA splint to ligate DNA *CONCLUSION: Need to check literature and/or call NEB but it seems SplintR has high ssDNA ligase activity, making it unusable for high specificity padlock probe applications **Checked and there is no reported ssDNA ligase activity, therefore it must be probes annealing to other probes **"Template-independent ligation" ===After meeting with Dr. Zhang=== *Reason PosCtrl does not show higher ligation is because forgot 95C is needed to denature gDNA! *He pointed out there is separation between NTC/30ng and 150ng/820ng PCR curves **Therefore there is some probe/splint ligation going on and conditions (eg temperature/time/concentration) need to be tuned to increase this separation ===Next Experiment=== *Find shortest amount of time necessary for SplintR Ligase at 37C **Minimize this time so less non-specific annealing of probes *Keep constant probe:target ratio for all *Try 3 NTCs with padlock probe concentrations matching 3 samples *Annealing temp: 95C 30sec -> Cool to 55C *Try different annealing times?
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