Brandon:LabNotes/Project1/2012-5-30: Difference between revisions
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*assume that taq polymerase is going bad since have freeze thawed many time in the past few weeks, will start using a new aliquot. Otherwise controls look fine. Also sample 8 is 1 uL of T7tspn-top2 from a previous IVT and has nice smear.. have lots of RNA to use for experimentation. | *assume that taq polymerase is going bad since have freeze thawed many time in the past few weeks, will start using a new aliquot. Otherwise controls look fine. Also sample 8 is 1 uL of T7tspn-top2 from a previous IVT and has nice smear.. have lots of RNA to use for experimentation. | ||
[[File:ZhangLab 2 2012-05-31 18hr 11min-labeled.jpg|600px]] | [[File:ZhangLab 2 2012-05-31 18hr 11min-labeled.jpg|600px]] | ||
*TBE gel after polyA tailing, RT, PCR | |||
*T7tspn-top3 for first 7 lanes. lane 8 is new primers on T7-tspn-top2 | |||
*backgound probably due to tranposon amplification? control looks like top3 custom transposon sample | |||
[[File:ZhangLab 2 2012-06-04 17hr 55min-labeled.jpg|600px]] | |||
*TBU gel after polyA tailing, RT, PCR | |||
*Looks like no large products amplified, is all junk. | |||
*Will try fragmentation methods and then polyA tailing, or could try denaturation | |||
[[File:ZhangLab 2 2012-06-04 19hr 24min-labeled.jpg|600px]] | |||
===polyA tailing (T7tspn-top3) Reference/Notes=== | ===polyA tailing (T7tspn-top3) Reference/Notes=== | ||
Line 250: | Line 264: | ||
**RNase III fragmentation (creates 5'-PO4 and 3'-OH ends on the fragments), then polyA addition. However cuts only dsRNA. | **RNase III fragmentation (creates 5'-PO4 and 3'-OH ends on the fragments), then polyA addition. However cuts only dsRNA. | ||
**chemical (Mg++, Zn++) fragmentation and then end repair with T4 polynucleotide kinase and ATP and polyA addition | **chemical (Mg++, Zn++) fragmentation and then end repair with T4 polynucleotide kinase and ATP and polyA addition | ||
**Rnase III versus Magnesium fragmentation profile comparisons, seems magnesium fragmentation gives more smaller fragments. Comparison below. | |||
[[File:E6150 v1 000014-mod.jpg|400px]] | |||
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*These methods leave different types of 3' and 5' ends. | *These methods leave different types of 3' and 5' ends. | ||
**Mg++ creates RNA fragments with 5' hydroxyl and 3' phosphate termini. Need to repair ends with T4 polynucleotide kinase and ATP, or Shrimp alakaline | **Mg++ creates RNA fragments with 5' hydroxyl and 3' phosphate termini. Need to repair ends with T4 polynucleotide kinase and ATP, or Shrimp alakaline phosphatase or antarctic phosphatase. Then can treat with polyA polymerase. | ||
**RNase III results in 2 bases 3' overhangs, and those ends contain 5'-PO4 and 3'-OH ends on the fragments. | **RNase III results in 2 bases 3' overhangs, and those ends contain 5'-PO4 and 3'-OH ends on the fragments. | ||
**Zn++ is assumed to produce ends like Mg++ since fragmentation uses a divalent metal ion and heat to fragmentt RNA | **Zn++ is assumed to produce ends like Mg++ since fragmentation uses a divalent metal ion and heat to fragmentt RNA | ||
Line 268: | Line 285: | ||
* | Methods used by different companies | ||
* | *NEB sells RNAase III kit for fragmenting mRNA | ||
** | *Ambion RNA protocol uses it for 100 ng samples in [http://products.invitrogen.com/ivgn/product/4454073 Ambion® RNA-Seq Library Construction Kit] and in their kit for "SOLiD™ Total RNA-Seq Kit". Also in "Ion Total RNA-Seq Kit". | ||
* | **do 5' and 3' ligation for small RNA kit, and for large, [http://www.umassmed.edu/uploadedFiles/nemo/Landing_Pages/UMASS_SOLiD%20Applications_4_12_10(1).pdf powerpoint presentation small RNA, transcript libraries] | ||
*Epicentre uses some sort of "chemical fragmentation" probably with Mg++ or Zn++. Uses random hexamers for amplification so don't need to have correct 5' and 3' ends for ligation. Also uses RNase III cutting of dsRNA for formation of siRNA. [http://www.epibio.com/item.asp?ID=395 RNase III, E. coli] | |||
** | *Illuminia uses RNA adaptor ligation for small RNAs with both 5' and 3' ends with this kit: [http://support.illumina.com/sequencing/sequencing_kits/truseq_small_rna_sample_prep_kit.ilmn small RNA sample prep kit]. Illuminia uses Mg++ chemical fragmentation protocol for normal RNA, and directional RNA-seq [http://support.illumina.com/sequencing/sequencing_kits/truseq_rna_sample_prep_kit_v2/compatibility.ilmn TruSeq RNA Sample Prep Kit v2 Support]. This was used in [http://www.nature.com/nature/journal/v471/n7336/full/nature09798.html Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells ] where 50 ng of RNA was isolated from cells to crate RNA-seq libraries. | ||
**first strand synthesis is with random hexamers and MMLV RT, second strand synthesis is dUTP synthesis with DNA polymerase I(E. coli), RNase H, and DNA Ligase. nick translation replacement of mRNA to synthesize second strand cDNA. Rnase H inserts and nicks RNA, providing 3' OH primers for DNA pol I. 5'-3 exonuclease acitivty of DNAP I removes DNA in the way of synthesis. DNA ligase links gaps to complete ds-cDNA strand. [http://cshprotocols.cshlp.org/content/2006/1/pdb.prot3560 Construction of cDNA Libraries (csh protocol)], [http://www.fermentas.com/en/products/all/molecular-cloning/kits/k2561-revertaid-premium-double-standed-cdna-synthesys-kit?print fermentas ReverAid ds-cDNA synthesis kit] | |||
Stage 2: Second-strand Synthesis | |||
*clontech does RT PCR off of polyA RNA, then fragmentation with covaris and end labeling.[http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/cDNA_Synthesis_Kits/Ultra_Low_Input_RNA_cDNA_Synthesis?sitex=10020:22372:US clontech SMARTer Ultra Low Input RNA for Illumina Sequencing] | |||
===rnase III=== | |||
Considerations to take into account for Rnase III fragmentation, and probably will produce dsRNA. | |||
*Invitrogen MEGAscript RNAi Kit is basically the same as MEGAscript T7 Kit | |||
**RNAi kit has more solutions/consumables, both contain same enzyme mix for T7 synthesis. Provides a method to create dsRNA which is highly similar to how the fragmentation library is being amplified, with two T7's on the ends of fragments. Either can be synthesized in sense and antisense direction in one reaction or in two different reactions. Below from MEGAscript RNAi kit manual. | |||
*[[File:Untitled.png|600px]] | |||
*Provides good template for RNase III which specifically cuts dsRNA. And shows this is already a used and well documented method in generation of siRNAs, but not for sequencing library amplification. | |||
*possible issues with dsRNA and higher Tm's? Espically since RNA-RNA and DNA-RNA structures have higher Tm's than DNA-DNA. paper showing some. [http://pubs.acs.org/doi/pdf/10.1021/bi00034a013 Relative Thermodynamic Stability of DNA, RNA, and DNA:RNA Hybrid Duplexes: Relationship with Base Composition and Structure] | |||
*RNase III fragmentation produces ends that are easy to ligate fragments to or use with polyA polymerase. End repair is not needed. | |||
*Only caveat is it seems to preferentially produce 25 ish bp fragments cutting from 5' and 3' ends. want to save 5' end since that is where the sequence tag is. | |||
Rnase III cuts from both 5' and 3' ends (for 21-25 bp fragments) | |||
*siRNA design for invitrogen kit places your sequence at the 5' end. | |||
*dicer is in the same family and makes same size nucleotide fragments | |||
*[http://www.nature.com/nature/journal/v475/n7355/abs/nature10198.html Dicer recognizes the 5′ end of RNA for efficient and accurate processing] | |||
*[http://products.invitrogen.com/ivgn/product/AM2290 invitrogen RNase III description] | |||
bc-RNase III | |||
*[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1935012/ Processing of Bacillus subtilis small cytoplasmic RNA: evidence for an additional endonuclease cleavage site] | |||
*[http://rnajournal.cshlp.org/content/6/8/1142.full.pdf Substrate recognition by a eukaryotic RNase III: the double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5'-AGNN-3' tetraloop.] | |||
*[http://www.ncbi.nlm.nih.gov/pubmed/9677377 Bacillus subtilis RNase III cleaves both 5'- and 3'-sites of the small cytoplasmic RNA precursor.] | |||
also showing random sites and 5' 3' sites | |||
*[http://www.pnas.org/content/74/3/984.full.pdf Nucleotide sequence surrounding a ribonuclease III processing site in bacteriophage T7 RNA] | |||
*RNase III only cuts dsRNA | |||
only dsRNA, not DNA-RNA: [http://www.jbc.org/content/249/4/1314.full.pdf Ribonuclease III Does Not Degrade Deoxyribonucleic Acid - Ribonucleic Acid Hybrids] | |||
NOT DNA-DNA, supercoiled DNA-DNA, ssRNA, doesn't remove phophates [http://www.neb.com/nebecomm/ManualFiles/manualE6146.pdf NEBNext® RNase III RNA Fragmentation Module] | |||
ssDNA?? | |||
VERY IMPORTANT!! can possibly block the 5' end by adding DNA primer prior to RNase III fragmentation to anneal and form DNA-RNA hybrid, since RNase III cannot degrade DNA-RNA, ssDNA or ssRNA strands. | |||
*This won't be an issue in downstream steps, including polyA addition, more specifically MMLV RT,and PCR. can add in 2x amount of forward primer during PCR since it will form primer dimer with first strand. | |||
* | |||
MMLV can displace short DNA sequences (strand displacement) | |||
[http://www.neb.com/nebecomm/products/productM0253.asp can transcribe across RNA- DNA junction, stated in FAQs] | |||
searched: "mmlv strand displacement" | |||
[http://tools.invitrogen.com/content/sfs/productnotes/F_071215_SuperScript%20II-TS-TL-MKT-HL.pdf Invitrogen superscript II FAQ] "MMLV and SuperScript™ will strand displace to a small extent (<100 nt, about 30 nucleotides)" | |||
===RNA fragmentation protocol (RNase III)=== | ===RNA fragmentation protocol (RNase III)=== | ||
Starting Material: Purified mRNA (50–250 nanograms) | Starting Material: Purified mRNA (50–250 nanograms) | ||
X uL Purified mRNA (50-250 nanograms) | 1. Add 5' end blocking DNA primer and do annealing to form DNA-RNA hybrid. | ||
*a. add 2 uL of blocking oligo (10 uM) to aliquot RNA sample that will be used in step 2. | |||
*b. Incubate at 95C for 2 minutes. | |||
*c. cool to RT at 0.1 C/s | |||
2. Mix the following components in a sterile PCR tube: | |||
X uL Purified mRNA + blocking primer (50-250 nanograms) | |||
1 uL RNase III (1 unit/μl) | 1 uL RNase III (1 unit/μl) | ||
2 uL RNase III Reaction Buffer (10X) | |||
2 uL Nuclease-Free Water | 2 uL Nuclease-Free Water | ||
add in DNA primer to protect 5' end since don't want degradation?? | |||
_______ | _______ | ||
20 uL total volume | 20 uL total volume | ||
3. Incubate in a preheated thermal cycler for 5 minutes at 37°C. | |||
4. Add 80 μl of cold Nuclease-Free water. (OR protease digestion of enzyme and inactivation of protease) | |||
5. Transfer tube to ice. | |||
6. Clean up RNA fragments using a RNA column purification kit or ethanol precipitation. |
Latest revision as of 01:34, 16 June 2012
Experiments needed for T7 transposon t7tspn-top3; RNA fragmentation info/plan[edit]
- modification to top portion of tspn so it can have a Tm of 62, so RT-PCR can be performed without a stem looping issue blocking binding of primers
- procotols from shendure paper, shendure paper transposition
1. annealing of ME sequence to T7 transposon sequence
- a. Make 100 uM stock solution of T7tspn-top3 and T7tspn-bot.
- b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL.
- c. cool to RT at 0.1 C/s
2. transposome complex generation, run controls!!!
- add the below components into one tube and incubate for 20 minutes at RT
1.25 uL of annealed transposon 1.25 uL of 100% sterile glycerol 2.50 uL of Ez-TN5 transposase
- store at -20, is good for a year
3. transposition reaction
Samples: 1. custom transposon1 (T7tspn-top3) 2. nextera normal 3. without transposome complex 4. no genomic DNA (has transposome complex) 5. Jurkat DNA only 6. annealed transposon only 7. Nuclease free H20 only
- add the below into one tube and incubate for 8 minutes at 55C.
1 uL nextera HMW buffer 1 uL genomic DNA (5 ng Jurkat DNA) 2 uL Nuclease free water 1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL) ___________ 5 uL total solution
method used in shendure paper: 4 uL nextera HMW buffer X uL genomic DNA at prepared quantities X uL Nuclease free water ______ 17.5 uL total solution add 2.5 uL of prepared transposomes
4. Protease digestion of transposase, protease inactivation
To each tube, add: 1 uL Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final []) Incubate: 50C 10 minutes, 70C 20 minutes
5. Fill in reaction
- Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
- IVT on filled in reaction and not filled in reaction in to show fill-in is working.
6. should now be ready for IVT.
Maxiscript (Ambion) T7 Protocol[edit]
DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
1. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) on ice, but keep 10X transcription buffer at room temp
2. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!!
bring to 20 uL with Nuclease free water X uL Nuclease free H2O X uL DNA template (list 1 ug) 2 uL 10X Transcription Buffer 1 uL 10 mM ATP 1 uL 10 mM CTP 1 uL 10 mM GTP 1 uL 10 mM UTP 2 uL T7 Enzyme Mix
3. Incubate reactions at 37C overnight. (>10 uM limiting nucleotide)
4. (Optional) Add 1 uL of TURBO DNase, mix well, incubate at 37C for 15 minutes.
5. (Optional) Add 1uL of 0.5 M EDTA to stop the reaction
6. Remove unincorporated nucleotides for quantitation. (ethanol precipitation or column precipitation). Can also run TBU gel to view sample and controls
Ethanol preciptation: 1. Add 30 uL water to DNase-I treated transcription reaction to bring the volume to 50 uL. 2. Add 3 volumes 10% EtOH, .1 volumes 3M NaOAc, and 1/300 volumes glycol Blue to the solution obtained. In this case: 5 uL NaOAc (mix after adding) 150 uL 100% EtOH 1 uL (1/10 dilution Glycol Blue) 3. Chill solution in -80 for 30 minutes, cool centrifuge to 4C 4. Spin at 4C for >15 minutes at max speed. Chill 75% EtOH. 5. Blue pellet should be visible, Discard supernatent. 6. Wash the pellet with cold 75% EtOH 7. To completely remove incorporated NTPs, resuspend the pellet in 50 uL nuclease free water and repeat steps 1-6. Otherwise resuspend with nuclease free water. 8. Store RNA at -80, in aliquots if desired. Aliquot in use can be at -20.
- Now can nanodrop for quanitation or run gel. fragment RNA, polyA tail addition, adapter addition to 5' and 3' ends.
protocol for and polyA tailing and RT-PCR (T7-top3)[edit]
PolyA addition
- enzymatics PolyA polymerase.
1. assemble reaction: 1 uL 10X polyA polymerase reaction buffer (or 2 uL 5X first strand buffer from SMART MMLV RT) 1 uL polyA enzyme 1 uL 10 mM ATP bring to 10 uL with RNA or w/e 2. Incubate at 37C for 10 minutes 3. Heat inactivate at 70C for 20 minutes. (rui and NEB)
single strand synthesis
- Followed protocol for SMART MMLV Reverse Transcriptase
20 uL reaction 1. Add 2.5 uL 20 uM primer stock to RNA sample. Brint to final volume of 11.5 uL with Nuclease free H2O (SMART CDS Primer II A this time) 2. heat the mixture to 70C fo 3 minutes. Immediately cool on ice. 3. Add the following to the reaction. 4 uL 5X first strand buffer 2 uL dNTP mix 2 uL 100 uM DTT .5 uL SMART MMLV RT and mix (ADD LAST!!!!!) 4. Incuvate at 42C for 60 minutes 5. Terminate the reaction by heating at 70C for 10 minutes
second strand synthesis (qPCR)
KAPA SYBR FAST qPCR mix X35 cycles 25 uL KAPA SYBR 4 uL primers, 1 uL F, 1 uL R (T7-top3-sss) and (PCR Reverse, Rui has) 1 uL H2O 20 uL DNA template (used whole RT reaction KAPA SYBR cycles: 98C 3min, (98C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever
- terminate when curves saturate
qiaquick cleanup
- run on gel or w/e
- run qiaquick to clean sample before performing qPCR.
- 6 enzymes from 6 different reactions in there already.
results[edit]
- T7-top3 had satisfactory fragmentation profile. Will try perform IVT, polyA tailing, RT, and PCR and see if quality smear is obtained.
- If quality smear obtained, then size selection, cloning and transformation for validation. Use blunt end kit for to ensure ligations into vector.
File:ZhangLab 2 2012-05-31 11hr 24min-labeled.jpg
- T7-top3 RNA qualitative results with TBU gel
- odd that needed 3 uL of sample to confirm presence of smear (barely visible with 1 uL sample)
- assume that taq polymerase is going bad since have freeze thawed many time in the past few weeks, will start using a new aliquot. Otherwise controls look fine. Also sample 8 is 1 uL of T7tspn-top2 from a previous IVT and has nice smear.. have lots of RNA to use for experimentation.
File:ZhangLab 2 2012-05-31 18hr 11min-labeled.jpg
- TBE gel after polyA tailing, RT, PCR
- T7tspn-top3 for first 7 lanes. lane 8 is new primers on T7-tspn-top2
- backgound probably due to tranposon amplification? control looks like top3 custom transposon sample
File:ZhangLab 2 2012-06-04 17hr 55min-labeled.jpg
- TBU gel after polyA tailing, RT, PCR
- Looks like no large products amplified, is all junk.
- Will try fragmentation methods and then polyA tailing, or could try denaturation
File:ZhangLab 2 2012-06-04 19hr 24min-labeled.jpg
polyA tailing (T7tspn-top3) Reference/Notes[edit]
- going to try A tailing with PolyA polymerase first then RT with clontech primer. "3' SMART CDS Primer II A" from SMARTer Ultra Low Input RNA for Illumina Sequencing kit
- 20 μl 200 μl 3’ SMART CDS Primer II A (12 μM) 5’–AAGCAGTGGTATCAACGCAGAGTACT(30)N-1N–3’ (N = A, C, G, or T; N-1 = A, G, or C). Also synthesizing my own (rui has) since theirs is modified 25 μl 250 μl IS PCR Primer (12 μM)* 5’– AAGCAGTGGTATCAACGCAGAGT –3’
PolyA tail addition
- using Enzymatics polyA polymerase
10X Poly(A) Polymerase Reaction Buffer (B7460): 500 mM Tris-HCl 2.5 M NaCl 100 mM MgCl2 pH 7.9 @ 25°C
first and second strand synthesis
first strand synthesis (MMLV RT) RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN CTGTCTCTTATACACATCTTAATGTCTCCCA(30) -3' 3'- N,N-1,T(30)CATGAGACGCAACTATGGTGACGAA 5' (clontech primer ) PCR/second strand synthesis *taq polymerase as 5' -> 3' exonuclease activity so will destroy primer in bottom strand synthesis *only issue is possible hairpin formation of ends since they are complementary, thus not allowing binding of T7-sssF2, but it will be seen. Tm of complemntary sequence. GGGAGACATTAAGATGTGTATAAGAGACAG Tm=60.71 Tm of primer is, AAGCAGTGGTATCAACGCAGAGT TM=61.02 *single insertions will not have a problem, this is only with adajacent insertions. (T7-top3-sss) 5'- GGGAGACATTAAGATGTGTATAAGAGACAG -3' 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNN(NNNNNN)CTGTCTCTTATACACATCTTAATGTCTCCCA(30)GTACTCTGCGTTGATACCACTGCTT -3' 3'- CCCTCTGTAATTCTACACATATTCTCTGTC(NNNNNN)NNNNNN GACAGAGAATATGTGTAGAATTACAGAGGGT(30)CATGAGACGCAACTATGGTGACGAA -5' 3'- GACAGAGAATATGTGTAGAATTACAGAGGG 3'- TGAGACGCAACTATGGTGACGAA -5' (T7-top3-sss) (T7-PCR-R) Needed primers: RT: 5’– AAGCAGTGGTATCAACGCAGAGTACT(30)N-1N –3’ (from clontech kit) 5’– AAGCAGTGGTATCAACGCAGAGT –3’ (T7-PCR-R) (Tm 61.02) 5'- GGGAGACATTAAGATGTGTATAAGAGACAG -3' (T7-top3-sss) (Tm 63.03)
RNA fragmentation[edit]
- can either try:
- RNase III fragmentation (creates 5'-PO4 and 3'-OH ends on the fragments), then polyA addition. However cuts only dsRNA.
- chemical (Mg++, Zn++) fragmentation and then end repair with T4 polynucleotide kinase and ATP and polyA addition
- Rnase III versus Magnesium fragmentation profile comparisons, seems magnesium fragmentation gives more smaller fragments. Comparison below.
- Currently are a couple of methods that are readily available from NEB, and are used in other protocols.
- Mg++ fragmentation
- RNase III fragmentation
- Zn++ fragmentation in some protocols
- shearing with covaris
- These methods leave different types of 3' and 5' ends.
- Mg++ creates RNA fragments with 5' hydroxyl and 3' phosphate termini. Need to repair ends with T4 polynucleotide kinase and ATP, or Shrimp alakaline phosphatase or antarctic phosphatase. Then can treat with polyA polymerase.
- RNase III results in 2 bases 3' overhangs, and those ends contain 5'-PO4 and 3'-OH ends on the fragments.
- Zn++ is assumed to produce ends like Mg++ since fragmentation uses a divalent metal ion and heat to fragmentt RNA
- Other RNAases create different ends.
- covaris is random
Methods used by different companies
- NEB sells RNAase III kit for fragmenting mRNA
- Ambion RNA protocol uses it for 100 ng samples in Ambion® RNA-Seq Library Construction Kit and in their kit for "SOLiD™ Total RNA-Seq Kit". Also in "Ion Total RNA-Seq Kit".
- do 5' and 3' ligation for small RNA kit, and for large, powerpoint presentation small RNA, transcript libraries
- Epicentre uses some sort of "chemical fragmentation" probably with Mg++ or Zn++. Uses random hexamers for amplification so don't need to have correct 5' and 3' ends for ligation. Also uses RNase III cutting of dsRNA for formation of siRNA. RNase III, E. coli
- Illuminia uses RNA adaptor ligation for small RNAs with both 5' and 3' ends with this kit: small RNA sample prep kit. Illuminia uses Mg++ chemical fragmentation protocol for normal RNA, and directional RNA-seq TruSeq RNA Sample Prep Kit v2 Support. This was used in Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells where 50 ng of RNA was isolated from cells to crate RNA-seq libraries.
- first strand synthesis is with random hexamers and MMLV RT, second strand synthesis is dUTP synthesis with DNA polymerase I(E. coli), RNase H, and DNA Ligase. nick translation replacement of mRNA to synthesize second strand cDNA. Rnase H inserts and nicks RNA, providing 3' OH primers for DNA pol I. 5'-3 exonuclease acitivty of DNAP I removes DNA in the way of synthesis. DNA ligase links gaps to complete ds-cDNA strand. Construction of cDNA Libraries (csh protocol), fermentas ReverAid ds-cDNA synthesis kit
Stage 2: Second-strand Synthesis
- clontech does RT PCR off of polyA RNA, then fragmentation with covaris and end labeling.clontech SMARTer Ultra Low Input RNA for Illumina Sequencing
rnase III[edit]
Considerations to take into account for Rnase III fragmentation, and probably will produce dsRNA.
- Invitrogen MEGAscript RNAi Kit is basically the same as MEGAscript T7 Kit
- RNAi kit has more solutions/consumables, both contain same enzyme mix for T7 synthesis. Provides a method to create dsRNA which is highly similar to how the fragmentation library is being amplified, with two T7's on the ends of fragments. Either can be synthesized in sense and antisense direction in one reaction or in two different reactions. Below from MEGAscript RNAi kit manual.
- File:Untitled.png
- Provides good template for RNase III which specifically cuts dsRNA. And shows this is already a used and well documented method in generation of siRNAs, but not for sequencing library amplification.
- possible issues with dsRNA and higher Tm's? Espically since RNA-RNA and DNA-RNA structures have higher Tm's than DNA-DNA. paper showing some. Relative Thermodynamic Stability of DNA, RNA, and DNA:RNA Hybrid Duplexes: Relationship with Base Composition and Structure
- RNase III fragmentation produces ends that are easy to ligate fragments to or use with polyA polymerase. End repair is not needed.
- Only caveat is it seems to preferentially produce 25 ish bp fragments cutting from 5' and 3' ends. want to save 5' end since that is where the sequence tag is.
Rnase III cuts from both 5' and 3' ends (for 21-25 bp fragments) *siRNA design for invitrogen kit places your sequence at the 5' end. *dicer is in the same family and makes same size nucleotide fragments *Dicer recognizes the 5′ end of RNA for efficient and accurate processing *invitrogen RNase III description bc-RNase III *Processing of Bacillus subtilis small cytoplasmic RNA: evidence for an additional endonuclease cleavage site *Substrate recognition by a eukaryotic RNase III: the double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5'-AGNN-3' tetraloop. *Bacillus subtilis RNase III cleaves both 5'- and 3'-sites of the small cytoplasmic RNA precursor. also showing random sites and 5' 3' sites *Nucleotide sequence surrounding a ribonuclease III processing site in bacteriophage T7 RNA
- RNase III only cuts dsRNA
only dsRNA, not DNA-RNA: Ribonuclease III Does Not Degrade Deoxyribonucleic Acid - Ribonucleic Acid Hybrids NOT DNA-DNA, supercoiled DNA-DNA, ssRNA, doesn't remove phophates NEBNext® RNase III RNA Fragmentation Module ssDNA??
VERY IMPORTANT!! can possibly block the 5' end by adding DNA primer prior to RNase III fragmentation to anneal and form DNA-RNA hybrid, since RNase III cannot degrade DNA-RNA, ssDNA or ssRNA strands.
- This won't be an issue in downstream steps, including polyA addition, more specifically MMLV RT,and PCR. can add in 2x amount of forward primer during PCR since it will form primer dimer with first strand.
MMLV can displace short DNA sequences (strand displacement) can transcribe across RNA- DNA junction, stated in FAQs searched: "mmlv strand displacement" Invitrogen superscript II FAQ "MMLV and SuperScript™ will strand displace to a small extent (<100 nt, about 30 nucleotides)"
RNA fragmentation protocol (RNase III)[edit]
Starting Material: Purified mRNA (50–250 nanograms)
1. Add 5' end blocking DNA primer and do annealing to form DNA-RNA hybrid.
- a. add 2 uL of blocking oligo (10 uM) to aliquot RNA sample that will be used in step 2.
- b. Incubate at 95C for 2 minutes.
- c. cool to RT at 0.1 C/s
2. Mix the following components in a sterile PCR tube:
X uL Purified mRNA + blocking primer (50-250 nanograms) 1 uL RNase III (1 unit/μl) 2 uL RNase III Reaction Buffer (10X) 2 uL Nuclease-Free Water add in DNA primer to protect 5' end since don't want degradation?? _______ 20 uL total volume
3. Incubate in a preheated thermal cycler for 5 minutes at 37°C.
4. Add 80 μl of cold Nuclease-Free water. (OR protease digestion of enzyme and inactivation of protease)
5. Transfer tube to ice.
6. Clean up RNA fragments using a RNA column purification kit or ethanol precipitation.