Brandon:LabNotes/Project1/2012-10-26
Custom transposon with IVT amplification, round 3[edit]
- will repeat IVT assay on 1000, 500, 100 cells, and nextera transposition on 150K, 50K, 10K, 1K, 500, and 100 cells.
- got positive results from round 2, showing IVT to be more efficient and better data for calling peaks can see here. Data from nextera seems to be dependent on the number of reads, regardless of cellular concentration, not sure why.
- experiment performed from round 2 will be repeated and will use the same cell line, GM12878. IVT round 2 experiment protocol
- to further show abilities and limitations of Nextera method, 10K, 50K and 150K cells will be used with nextera tagmentation protocol. Increased amounts of nextera transposomes will be used with each cellular amount since it has been observed lower cellular concentrations have better nextera transposome integration with higher transposomes/cells ratio.
- procotols from shendure paper, shendure paper transposition
- using T7-top2 for transposition reactions t7tspn-top2
- Improvements from last assay
- GM12878 DNA will be purified from GM12878 using RNase A (or Rnase mixture) and Protease AND proteinase K. Highly accessible regions are still highly accessible in pure DNA samples, want want to ensure proteins are not bound and RNA is not interfering. If those regions are still accessible then dunno why..
Why using RNAase III for fragmentation
- Rnase III creates 5'-PO4 and 3'-OH termini on the fragments
- however, Cuts ONLY dsRNA (used DNA blocking primer to block that)
- Preferentially cuts from 5’ and 3’ ends (used blocking primer)
- Rnase III results in 2 base 3’ overhangs
- Used in the generation of siRNAs for knockdown
- Mg++ fragmentation creates 5' OH and 3' PO4 termini, thus have to do end repair.
- End Repair with Shrink Akaline phosphatase, PNK, Antarctic phosphatase
- random nonamer is less selective and can prime off of more sequences
buffers compositions[edit]
1X T7 buffer: 400 mM Tris Hcl 8 mM MgCl2 2 mM spermidine-Hcl 25 mM NaCl PH 7.9 1X NEBNext RNase III Reaction Buffer: 10 mM Tris-HCl 10 mM Mg(Cl)2 1 mM DTT 60 mM NaCl pH 8.3 @ 25°C 10X Poly(A) Polymerase buffer: 500 mM Tris-HCl 2.5 M NaCl 100 mM MgCl2 pH 7.9 @ 25°C MMLV Invitrogen (though using clontech) 5X First-Strand Buffer 250 mM Tris-HCl (pH 8.3 at room temperature 375 mM KCl 15 mM MgCl2 0.1 M DTT
Before starting protocols[edit]
1. Check if have enough reagents etc for the protocol
- lysis buffer
- nextera transposomes
- transposase/transposome
- IVT reaction mixture
- cells etc
- blocking primer, RNase III, PolyA Polymerase, ATP, taq polymerase, other primers
2. Purify GM12878 DNA from GM12878 cells with DNeasy blood and tissue kit for cells (USE PROTEINASE K (invitrogen) AND protease (qiagen) AND RNAase A). quanititate DNA with nanodrop.
GM12878 DNA purification method used (followed DNeasy kit protocol for washes etc): a. pellet cells b. resuspend in: 1. 100 ul PBS 2. 10 uL proteinase K 3. 3 uL 5 AU Qiagen protease 4. 4 uL A797A promega RNase solution (RNase A) c. add 200 uL buffer AL, incuabate at 56C for 10 minutes d. step 3 in DNeasy kit protocol until finish
3. samples
Samples: 1. 1000 cells IVT 2. 500 cells IVT 3. 100 cells IVT 4. 6 ng purified DNA IVT 5. 3 ng purified DNA IVT 6. 600 pg purified DNA IVT 7. 150K cells nextera tagmentation 8. 50K cells nextera tagmentation 9. 10K cells nextera tagmentation 10. 1000 cells nextera tagmentation 11. 500 cells nextera tagmentation 12. 100 cells nextera tagmentation 13. 60 ng purified DNA nextera tagmentation 14. 6 ng purified DNA nextera tagmentation 15. 3 ng purified DNA nextera tagmentation 16. 600 pg purified DNA nextera tagmentation 17. 1000 cells lysed without transposome complex IVT 18. pure DNA only (6 ng) IVT 19. Nuclease free H20 only IVT 20. 1000 cells lysed without transposome complex nextera tagmentation 21. pure DNA only (6 ng) nextera tagmentation 22. Nuclease free H20 only nextera tagmentation
Nextera Tagmentation Protocol[edit]
1. See step 3 in IVT protocol. Do the cell lysis section for the "nextera tagmentation samples", samples 7-12,20,22.
2. Perform tagmentation on lysed cells and pure DNA. with 1:10 diluted nextera enzyme. (1:50 diluted in reaction). adjust amount of transposome on cell number
cells | ng DNA | nxta enzme to use |
150,000 | 180 | 3 uL not diluted |
50,000 | 120 | 2 uL not diluted |
10,000 | 60 | 1 uL not diluted |
1,000 | 6 | 3 uL 1/5 dltd enz |
500 | 3 | 2 uL 1/5 dltd enz |
100 | 0.6 | 1 uL, 1/10 dltd enz |
60 ng pure | 60 | 1 uL not diluted |
6 ng pure | 6 | 1 uL, 1/10 dltd enz |
3 ng pure | 3 | 1 uL, 1/10 dltd enz |
600 pg pure | 0.6 | 1 uL, 1/10 dltd enz |
Dilute nextera enzyme as specified above. For each rxn, used mix of: 1ul 5x LMW Buffer 2ul cell lysate Xul diluted enzyme Xul H2O ---------------------------- 5ul total / reaction 55C 10 min
3. Protease (Qiagen) digestion of transposition reactions.
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
4. First step of 2-step PCR thermocycling, terminate curves before saturation.
6 ul Tagmentation reaction 25 ul KAPA SYBR FAST qPCR mix 1 ul Orange Primer (10uM) 1 ul Blue Primer (10uM) 0.5 ul Bst Pol (5U/ul) 16.5 ul H2O ---------------- 50 uL total Orange primer CCTTGCCAGCCCGCTCAG 18nt Blue primer CCTCCCTCGCGCCATCAG 18nt PCR cycling. 72C for 3 minutes is for gap fill in. 72C 3min -> 95C 30 sec -> (95C 10sec -> 58C 30 sec -> 72 3min) x 25 -> 72C 3min. Monitor the reactions on a real-time thermal cycler and terminate them before the curves reach saturation.
5. Begin 2-Step AMPure beads purification to remove primers, add index primers, for low input. (with forward read primer and reverse index primers)
a. see 2-step AMpure beads protocol, start from beginning for first purification b. After cleaning, second PCR reaction to add index primers Barcodes to use: Index 55, NX adaptor 7. 150K cells nextera tagmentation Index 56, NX adaptor 8. 50K cells nextera tagmentation Index 57, NX adaptor 9. 10K cells nextera tagmentation Index 58, NX adaptor 10. 1000 cells nextera tagmentation Index 59, NX adaptor 11. 500 cells nextera tagmentation Index 60, NX adaptor 12. 100 cells nextera tagmentation Index 61, NX adaptor 13. 60 ng purified DNA nextera tagmentation Index 62, NX adaptor 14. 6 ng purified DNA nextera tagmentation Index 63, NX adaptor 15. 3 ng purified DNA nextera tagmentation Index 64, NX adaptor 16. 600 pg purified DNA nextera tagmentation Index 68, NX adaptor 20. 1000 cells lysed without transposome complex nextera tagmentation Index 69, NX adaptor 21. pure DNA only (6 ng) nextera tagmentation Index 70, NX adaptor 22. Nuclease free H20 only nextera tagmentation
add the following to above dry tube: 25 uL KAPA HF mix (used KAPA SYBR FAST qPCR mix instead) 1 uL adaptor1 1 uL of adaptor2 (barcode) 23 uL nuclease free H2O c. PCR cycling for addition of index primer 5 cycles 72C 3min -> 95C 30 sec -> (95C 10sec -> 60C 30 sec -> 72 1 min) x 2 (95C 10 sec -> 62C 30 sec -> 72C 1 min) X 3 -> 72C 3min -> 4C forever d. second phase of AMpure beads 2-step beads purification.
- can run on gel and check smears to see for library.
- should be ready for sequencing now?
IVT Protocol[edit]
- If need to make more transposome, do first 2 steps.
1. annealing of ME sequence to T7 transposon sequence
- a. Make 100 uM stock solution of T7tspn-top2 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. Prepare samples, lyse cells with lysis buffer
use pure GM12878 DNA
Samples: 1. 1000 cells IVT 2. 500 cells IVT 3. 100 cells IVT 4. 6 ng purified DNA IVT 5. 3 ng purified DNA IVT 6. 600 pg purified DNA IVT 17. 1000 cells lysed without transposome complex IVT 18. pure DNA only (6 ng) IVT 19. Nuclease free H20 only IVT
- Make 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.
- Prepare aliquots of lymphocytes GM12878 (have GM20431, GM12878, MEFs) that contain 1000, 500, 100 cells.
- spin down cells to concentrate them as necessary.
- Prepare 2X LB from 10X buffer. mineral oil optional.
- if needed make 4 or 6 uL solutions. keep 1:1 ratio of cells:lysis buffer
- make each cellular concentration in triplicate for AluI amplification with PCR for cell number quantification! store samples and perform quantification later. Use pure DNA nanodropped controls!!
AluI PCR quant 5 uL AluI 245-263 primer 5 uL AluI 21-40 primer X sample X water _________ 25 uL
- incubate below mixtures at 37C for 30 mins.
' | 1. 1K IVT | 2. 500 IVT | 3. 100 IVT | 7. 150K nxta | 8. 50K nxta | 9. 10K nxta | 10. 1K nxta | 11. 500 nxta | 12. 100 nxta | 17. 1K IVT con | 19. NTC IVT | 20. 1K nxta con | 22. NTC nxta |
cells | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul PBS | 1 ul | 1 ul PBS |
2X LB | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul |
4. transposition reaction, using (T7tspn-top2)
- add the below into one tube and incubate for 10 minutes at 55C.
1 uL nextera LMW buffer 2 uL lysed/pure genomic DNA (X ng/pg DNA) 1.2 uL Nuclease free water .8 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
5. 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
6. Fill in reaction
- Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
7. Maxiscript (Ambion) T7 Protocol, IVT
- DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) on ice, but keep 10X transcription buffer at room temp b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!! bring to 20 uL with Nuclease free water 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 b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide)
8. Clean RNA with with Zymo RNA Clean & Concentrator-5 Kit
- can quantitate with Qubit if needed, and run TBU-gel
9. Perform RNase III fragmentation (NEB):
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. use T7-frag-block-top2 for T7-top2. T7-frag-block for T7-top3 *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) .5 uL RNase III (1 unit/μl) 1 uL RNase III Reaction Buffer (10X) 5.5 uL Nuclease-Free Water add in DNA primer to protect 5' end since don't want degradation?? _______ 10 uL total volume 3. Incubate in a preheated thermal cycler for 5 minutes at 37°C. 4. Transfer tube to ice.
10. Zymo RNA clean and concentrator cleanup.
- Resuspend in appropriate volume in nuclease free H2O (12 uL last time)
- not doing protease digestion after RNA fragmentation since seems it wasn't as effective in previous RNase III fragmentation
11. Poly(A) Addition with polyA polymerase (Enzymatics)
- enzymatics PolyA polymerase.
a. assemble reaction: 2 uL 5X SMART MMLV first strand buffer (rather than 10X polyA buffer) 1 uL polyA enzyme 1 uL 10 mM ATP bring to 10 uL with RNA or w/e b. Incubate at 37C for 10 minutes c. Heat inactivate at 70C for 20 minutes. (rui and NEB)
12. single strand synthesis MMLV RT (Clontech)
- Followed protocol for SMART MMLV Reverse Transcriptase
20 uL reaction 1. Add 2.5 uL 20 uM primer stock to RNA sample. Bring to final volume of 12.5 uL with Nuclease free H2O (one from BENG160 class, T20VN_PE_R) 2. heat the mixture to 70C fo 3 minutes. Immediately cool on ice. 3. Add the following to the reaction. 2 uL 5X first strand buffer 2 uL dNTP mix 2 uL 100 uM DTT 1 uL N-H20 .5 uL SMART MMLV RT and mix (ADD LAST!!!!!) ____ 20 uL total 4. Incuvate at 42C for 60 minutes 5. Terminate the reaction by heating at 70C for 10 minutes
13. second strand synthesis (qPCR) (KAPA), addition of barcodes
Samples: Index 49, N2 adaptor 1. 1000 cells IVT Index 50, N2 adaptor 2. 500 cells IVT Index 51, N2 adaptor 3. 100 cells IVT Index 52, N2 adaptor 4. 6 ng purified DNA IVT Index 53, N2 adaptor 5. 3 ng purified DNA IVT Index 54, N2 adaptor 6. 600 pg purified DNA IVT Index 65, N2 adaptor 17. 1000 cells lysed without transposome complex IVT Index 66, N2 adaptor 18. pure DNA only (6 ng) IVT Index 67, N2 adaptor 19. Nuclease free H20 only IVT
KAPA SYBR FAST qPCR mix until saturation, X35 cycles 25 uL KAPA SYBR FAST qPCR mix 4 uL primers, 2 uL F, 2 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX]) 1 uL H2O 20 uL DNA template (use whole RT reaction) KAPA SYBR cycles: 98C 3min, (98C for 30s, 60C for 30s, 72C for 2 min) X35, 72C for 5 min, 4C forever
- terminate before curves saturate (usually cycle 6-7)
14.qiaquick cleanup
- can quanitate with nanodrop
15. Gel Size selection
- gel size select from 400-800 bp, follow gel size selection protocol
- do not need to include controls.
16. Cloning and Transformation, then genewiz sequencing for verification of inserts
17. Submit for sequencing if genewiz sequencing checks out.
Results[edit]
Nextera results and gels
- nanodrop results for nextera samples
# | sample | DNA | ' |
7 | 150K cells nextera tagmentation | 111.85 | ng/ul |
8 | 50K cells nextera tagmentation | 165.6 | ng/ul |
9 | 10K cells nextera tagmentation | 169.59 | ng/ul |
10 | 1000 cells nextera tagmentation | 117.73 | ng/ul |
11 | 500 cells nextera tagmentation | 94.03 | ng/ul |
12 | 100 cells nextera tagmentation | 57.09 | ng/ul |
13 | 60 ng purified DNA nextera tagmentation | 128.13 | ng/ul |
14 | 6 ng purified DNA nextera tagmentation | 101.87 | ng/ul |
15 | 3 ng purified DNA nextera tagmentation | 85.11 | ng/ul |
16 | 600 pg purified DNA nextera tagmentation | 93.49 | ng/ul |
20 | 1000 cells lysed without tspsome nextera tagmentation | 11.68 | ng/ul |
21 | pure DNA only (6 ng) nextera tagmentation | 14.33 | ng/ul |
22 | Nuclease free H20 only nextera tagmentation | 23.32 | ng/ul |
- ran gel after 2 step AMPure beads purification (addition of illuminia primers, barcodes)
File:ZhangLab 2 2012-10-31 15hr 50min-labeled.jpg File:ZhangLab 2 2012-10-31 15hr 54min-labeled.jpg
- qPCR curves for blue/orange amplification of products
- all products PCR amplification curves
- samples in order
Samples: 7. 150K cells nextera tagmentation 8. 50K cells nextera tagmentation 9. 10K cells nextera tagmentation 10. 1000 cells nextera tagmentation 11. 500 cells nextera tagmentation 12. 100 cells nextera tagmentation 13. 60 ng purified DNA nextera tagmentation 14. 6 ng purified DNA nextera tagmentation 15. 3 ng purified DNA nextera tagmentation 16. 600 pg purified DNA nextera tagmentation 20. 1000 cells lysed without transposome complex nextera tagmentation 21. pure DNA only (6 ng) nextera tagmentation 22. Nuclease free H20 only nextera tagmentation
File:2012-10-30 round 3 accessibility nextera orange blue amplification.bmp
- only nextera treated accessibility samples, amplification curves
- samples in order
Samples: 7. 150K cells nextera tagmentation 8. 50K cells nextera tagmentation 9. 10K cells nextera tagmentation 10. 1000 cells nextera tagmentation 11. 500 cells nextera tagmentation 12. 100 cells nextera tagmentation
File:2012-10-30 round 3 accessibility nextera orange blue amplification cells acc.bmp
- only nextera treated pure DNA samples and controls, amplification curves
- samples in order
Samples: 13. 60 ng purified DNA nextera tagmentation 14. 6 ng purified DNA nextera tagmentation 15. 3 ng purified DNA nextera tagmentation 16. 600 pg purified DNA nextera tagmentation 20. 1000 cells lysed without transposome complex nextera tagmentation 21. pure DNA only (6 ng) nextera tagmentation 22. Nuclease free H20 only nextera tagmentation
File:2012-10-30 round 3 accessibility nextera orange blue amplification pure dna, controls.bmp
IVT amplification results/gels
- Used 1 uL for TBU gel, 1 uL for Qubit quantitation of RNA. RNA eluted in 14 uL N-H20 total.
' | [sample] | total ng |
1. 1000 cells IVT | 20 ng/ul | 280 ng |
2. 500 cells IVT | 25.1 ng/ul | 351.4 ng |
3. 100 cells IVT | 37.3 ng/ul | 522.2 ng |
4. 6 ng purified DNA IVT | 61.5 ng/ul | 861 ng |
5. 3 ng purified DNA IVT | 60.9 ng/ul | 852.6 ng |
6. 600 pg purified DNA IVT | 60.4 ng/ul | 845.6 ng |
17. 1000 cells lysed w/out transposome complex IVT | Out of Range | Out of Range |
18. pure DNA only (6 ng) IVT | Out of Range | Out of Range |
19. Nuclease free H20 only IVT | Out of Range | Out of Range |
- graph of ng RNA versus cell number for IVT RNA amplification. for accessibility samples gave opposite of expected results, not sure why. possible containment? dunno. regardless still gave satisfactory amounts of RNA from IVT amplification, slightly less then accessibility round2. possible that freezer (-20) defreeze had an effect.
File:Picture1-round3-qubit.png
- RNA after IVT amplification, TBU gel. Expected more RNA in accessibility samples but still satisfactory total amounts.
File:ZhangLab 2 2012-10-31 17hr 11min-labeled.jpg
- qPCR curves for barcode/adaptor addition
- not sure what happened. diluted SYBR green from 10,000X to 250X and added to KAPA robust PCR mix, which didn't have SYBR green initially. Where first reading started out is what is expected based on cell number/DNA amounts in the samples. However ran gel and looked fine
Samples: 1. 1000 cells IVT 2. 500 cells IVT 3. 100 cells IVT 4. 6 ng purified DNA IVT 5. 3 ng purified DNA IVT 6. 600 pg purified DNA IVT 17. 1000 cells lysed without transposome complex IVT 18. pure DNA only (6 ng) IVT 19. Nuclease free H20 only IVT
File:2012-10-30 round 3 accessibility IVT barcodes.bmp
- TBE gel after barcode/illuminia adaptor addition. TBE gel, 50 uL sample total
fragments above 127 bp will have an insert, at least 157 for good insert 5’ end GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG 3’ end T20VN_PE_R AAAAAAAAAAAAAAAAAAAAACTAGCCTTCTCGCCAAGTCGTCCTTACG 3’ end of PCR_R.N2Ind[XX] with ILA adaptor GCTCGAACATTAGAGCATACGGCAGAAGACGAAC
File:ZhangLab 2 2012-11-01 16hr 43min-labeled.jpg
- TBU gel after barcode/illuminia adaptor addition. TBE gel had thick bands so wanted to make sure it was still right sized fragments and not DNA hairballs.
File:ZhangLab 2 2012-11-01 18hr 02min-labeled.jpg
- gel size selection
File:ZhangLab 2 2012-11-09 11hr 33min.jpg
- validating gel size selection
File:ZhangLab 2 2012-11-09 15hr 32min-labeled.jpg
- cloning and transformation results
- shown to be uninformative. samples that were successful in last run had bands between 400-600 after PCR amplification. was expected this was not working based on previous gels and barcodes addition results
- has newly annealed trasposome in lane 10 only to check.
File:ZhangLab 2 2012-11-13 15hr 15min-labeled.jpg File:ZhangLab 2 2012-11-13 15hr 33min-labeled.jpg File:ZhangLab 2 2012-11-13 15hr 34min-labeled.jpg
conclusions
- seems like something went wrong since all the curves went down during PCR and the gels did not look corrected. Expected to see different amounts based on input DNA.
- IVT samples did not amplify as much as [round2 IVT data]. when comparing TBU gel smears they amplified alot more in round2. Possible reasons are the -20 defreeze over the weekend (since someone left the door open) could have left the IVT kits and taq polymerases etc thawed for more than a day. Could also be cell lysis did not occur as efficiently.
- ordered new IVT kit and taq polymerases. will remake cell lysis buffer when performing experiment again.
- kun mentioned Rui got the same type of smears before and did cloning and transformation on them, and they did not turn out to well. Probably the same issue here
- Nextera samples worked fine and gave expected results. Will keep them for next sequencing run for when IVT samples work correctly.
- cloning and transformation uninformative. showed that didnt work.
Notes ETC[edit]
- since previous frag block was designed for T7-top3.
T7-frag-block-top2 5'- CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGATCTCCC/3InvdT/ Tm= 81.45
- Effeciency of DNase I to degrade ssDNA and DNA:RNA hybrids is 500X less effecient, according to Invitrogen site thus use longer incubation?
Barcodes used:
Samples: Index 49, N2 adaptor 1. 1000 cells IVT Index 50, N2 adaptor 2. 500 cells IVT Index 51, N2 adaptor 3. 100 cells IVT Index 52, N2 adaptor 4. 6 ng purified DNA IVT Index 53, N2 adaptor 5. 3 ng purified DNA IVT Index 54, N2 adaptor 6. 600 pg purified DNA IVT Index 55, NX adaptor 7. 150K cells nextera tagmentation Index 56, NX adaptor 8. 50K cells nextera tagmentation Index 57, NX adaptor 9. 10K cells nextera tagmentation Index 58, NX adaptor 10. 1000 cells nextera tagmentation Index 59, NX adaptor 11. 500 cells nextera tagmentation Index 60, NX adaptor 12. 100 cells nextera tagmentation Index 61, NX adaptor 13. 60 ng purified DNA nextera tagmentation Index 62, NX adaptor 14. 6 ng purified DNA nextera tagmentation Index 63, NX adaptor 15. 3 ng purified DNA nextera tagmentation Index 64, NX adaptor 16. 600 pg purified DNA nextera tagmentation Index 65, N2 adaptor 17. 1000 cells lysed without transposome complex IVT Index 66, N2 adaptor 18. pure DNA only (6 ng) IVT Index 67, N2 adaptor 19. Nuclease free H20 only IVT Index 68, NX adaptor 20. 1000 cells lysed without transposome complex nextera tagmentation Index 69, NX adaptor 21. pure DNA only (6 ng) nextera tagmentation Index 70, NX adaptor 22. Nuclease free H20 only nextera tagmentation
- N2 adaptor with illuminia adatpor on the 5' end.
My modifications for T7-tspns for 5' addition primers To use with (T7-top and T7-top2) transposons 5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’ (Tm=86.59) ILA adaptor blue Top2-5’end (T7-top2-PCR-iaf) Tm=66.79 (can be used if top1 is used for transposition)
- N2 adaptors with barcodes and illuminia adaptors (N9_PE_R and T20VN_PE_R)
Barcode ID | Barcode | Primer | Primer Name |
Ind49 | ACACAG | CAAGCAGAAGACGGCATACGAGATACACAGCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind49 |
Ind50 | AAAGGT | CAAGCAGAAGACGGCATACGAGATAAAGGTCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind50 |
Ind51 | GCGATA | CAAGCAGAAGACGGCATACGAGATGCGATACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind51 |
Ind52 | CGTGTC | CAAGCAGAAGACGGCATACGAGATCGTGTCCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind52 |
Ind53 | GTAGAA | CAAGCAGAAGACGGCATACGAGATGTAGAACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind53 |
Ind54 | GGACGT | CAAGCAGAAGACGGCATACGAGATGGACGTCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind54 |
Ind55 | AGTCGA | CAAGCAGAAGACGGCATACGAGATAGTCGACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind55 |
Ind56 | GTCTGA | CAAGCAGAAGACGGCATACGAGATGTCTGACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind56 |
Ind57 | GAAGGA | CAAGCAGAAGACGGCATACGAGATGAAGGACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind57 |
Ind58 | ATGCTG | CAAGCAGAAGACGGCATACGAGATATGCTGCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind58 |
Ind59 | TCTATC | CAAGCAGAAGACGGCATACGAGATTCTATCCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind59 |
Ind60 | ATCTGT | CAAGCAGAAGACGGCATACGAGATATCTGTCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind60 |
Ind61 | ATAGAG | CAAGCAGAAGACGGCATACGAGATATAGAGCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind61 |
Ind62 | GCTAAA | CAAGCAGAAGACGGCATACGAGATGCTAAACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind62 |
Ind63 | ACCAGG | CAAGCAGAAGACGGCATACGAGATACCAGGCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind63 |
Ind64 | CCAACT | CAAGCAGAAGACGGCATACGAGATCCAACTCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind64 |
Ind65 | AAGGAA | CAAGCAGAAGACGGCATACGAGATAAGGAACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind65 |
Ind66 | CCTCCA | CAAGCAGAAGACGGCATACGAGATCCTCCACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind66 |
Ind67 | CACGTC | CAAGCAGAAGACGGCATACGAGATCACGTCCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind67 |
Ind68 | CATAAC | CAAGCAGAAGACGGCATACGAGATCATAACCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind68 |
Ind69 | CCATAT | CAAGCAGAAGACGGCATACGAGATCCATATCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind69 |
Ind70 | GAAGTC | CAAGCAGAAGACGGCATACGAGATGAAGTCCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind70 |
Ind71 | CAAAGA | CAAGCAGAAGACGGCATACGAGATCAAAGACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind71 |
Ind72 | TGGCAG | CAAGCAGAAGACGGCATACGAGATTGGCAGCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind72 |
- nextera Adaptor 1, matches blue primer, CCTCCCTCGCGCCATCAG, 18nt
Adaptor 1*: 5′-AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG-3′
- nextera barcoded adaptor 2's and illuminia adaptors. matches orange primer, Orange primer CCTTGCCAGCCCGCTCAG, 18nt
Barcode ID | Barcode | Primer |
Ind49 | ACACAG | CAAGCAGAAGACGGCATACGAGATACACAGCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind50 | AAAGGT | CAAGCAGAAGACGGCATACGAGATAAAGGTCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind51 | GCGATA | CAAGCAGAAGACGGCATACGAGATGCGATACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind52 | CGTGTC | CAAGCAGAAGACGGCATACGAGATCGTGTCCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind53 | GTAGAA | CAAGCAGAAGACGGCATACGAGATGTAGAACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind54 | GGACGT | CAAGCAGAAGACGGCATACGAGATGGACGTCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind55 | AGTCGA | CAAGCAGAAGACGGCATACGAGATAGTCGACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind56 | GTCTGA | CAAGCAGAAGACGGCATACGAGATGTCTGACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind57 | GAAGGA | CAAGCAGAAGACGGCATACGAGATGAAGGACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind58 | ATGCTG | CAAGCAGAAGACGGCATACGAGATATGCTGCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind59 | TCTATC | CAAGCAGAAGACGGCATACGAGATTCTATCCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind60 | ATCTGT | CAAGCAGAAGACGGCATACGAGATATCTGTCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind61 | ATAGAG | CAAGCAGAAGACGGCATACGAGATATAGAGCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind62 | GCTAAA | CAAGCAGAAGACGGCATACGAGATGCTAAACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind63 | ACCAGG | CAAGCAGAAGACGGCATACGAGATACCAGGCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind64 | CCAACT | CAAGCAGAAGACGGCATACGAGATCCAACTCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind65 | AAGGAA | CAAGCAGAAGACGGCATACGAGATAAGGAACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind66 | CCTCCA | CAAGCAGAAGACGGCATACGAGATCCTCCACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind67 | CACGTC | CAAGCAGAAGACGGCATACGAGATCACGTCCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind68 | CATAAC | CAAGCAGAAGACGGCATACGAGATCATAACCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind69 | CCATAT | CAAGCAGAAGACGGCATACGAGATCCATATCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind70 | GAAGTC | CAAGCAGAAGACGGCATACGAGATGAAGTCCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind71 | CAAAGA | CAAGCAGAAGACGGCATACGAGATCAAAGACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind72 | TGGCAG | CAAGCAGAAGACGGCATACGAGATTGGCAGCGGTCTGCCTTGCCAGCCCGCTCAG |
Read primers used for sequencing:
T7tspn-Read1 and Nextera Read 1 primer are exactly the same except for first nucleotide on 5' end. for IVT generated samples: T7tspn-Read1 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' N2RevSeq2 (read primer 2) 5'- CTCGGCATTCCTGCTGAACCGCTCTTCCGATCT -3' N2IndSeq 5'- AAGAGCGGTTCAGCAGGAATGCCGAG -3' for Nextera generated samples: Nextera Read 1 Primer: 5′- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3′ Nextera Read 2 primer: 5′- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3′ Nextera Index Read Primer: 5′-CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG-3′
Stuff used for RNA-seq for BENG160 class
5’ end addition primers TSO_N10_BC[XX] [AAGCAGTGGTATCAACGCAGAGdUdU]NNNNNNNNNNTTTAGGrGrGrG adatpor1 5'- AAG CAG TGG TAT CAA CGC AGA G/ideoxyU//ideoxyU/ NNN NNN NNN NTT TAG GrGrGrG -3' P1-STRT 5’- [AATGATACGGCGACCACCGA][GATCT][AAGCAGTGGTATCAACGCAGAGT] -3’ (Tm=82.59) ILA adaptor blue Tm=64.31 adaptor1 Tm=61.02 STRT-SEQ 5'- [GATCT][AAGCAGTGGTATCAACGCAGAGTT] -3' adaptor1
3’ end addition primers T20VN_PE_R 5'-Bio-[GCATTCCTGCTGAACCGCTCTT]CCGATCTTTTTTTTTTTTTTTTTTTTTVN -3’ (Tm=78.92) adaptor2 Tm=65.68 PCR_R.N2Ind[XX] (XX= index number) 5’- [CAAGCAGAAGACGGCATACGAGAT][TACAAG]CTCG][GCATTCCTGCTGAACCGCTCTT] -3’ (Tm=87.12) ILA adaptor orange bc adaptor2 Tm=65.68
My modifications for T7-tspns for 5' addition primers To use with (T7-top and T7-top2) transposons 5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’ (Tm=86.59) ILA adaptor blue Top2-5’end (T7-top2-PCR-iaf) Tm=66.79 (can be used if top1 is used for transposition) To use with (T7-top3) transposon 5’- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3’ (Tm=81.40) ILA adaptor blue Top3-5’end (T7-top3-PCR-iaf) Tm=60.71