Brandon:LabNotes/Project1/2012-8-6
Custom transposon with IVT amplification, round 2
- comparing custom transposon to nextera tagmentation more effectively since using the same cell line, GM12878. Before nextera tagmentation used GM20431 cells.
- also using with proper controls and pure DNA.
- procotols from shendure paper, shendure paper transposition
- using T7-top2 for transposition reactions t7tspn-top2
- goal is to compare efficiency of DNA accessibility across different cellular concentrations. Also to test efficiency of tagmentation with custom transposon/transposome on pure DNA, and efficiency of amplification from IVT.
- Using GM12878 cells.
- Using mouse MEFs to check for possibility of contamination. want to obtain same type of results with custom transposon/IVT amplifiation as obtained in the previous accessibilty assay performed
- Improvments from last assay
- DNase used to remove transposons and blocking primer after IVT and fragmentation
- Use Rnase III for fragmentation. Random nomaer RT has more possible binding locations
- Cleaning RNA with Zymo RNA cleaning kit
UCSC genome browser mouse hypersensitivity tracts
- is present on the UCSC Genome Browser on Mouse July 2007 (NCBI37/mm9) Assembly
- DNaseI Hypersensitivity by Digital DNaseI from ENCODE/University of Washington
- could be helpful to do comparisons against but a few issues.
- immortalized cell lines were generated in house by the lab performing the study and are not buyable off of ATCC
- other cells lines are harvested from mice at 8 weeks of age, such as cells from the cerebellum or B cell lymphocytes.
- embryonic cells harvested and also had accessibiity performed on them.
- Thus can make comparisons from my analysis with MEFs but don't know how comparable they will be to UCSC UW DNase I Hypersensitivity tracts.
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
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
Protocol
- have enough?
- IVT reaction mixture
- transposase/transposome
- cells etc
- blocking primer, RNase III
- 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. 600 pg purified DNA IVT 6. 60 pg purified DNA IVT 7. 1000 cells nextera tagmentation 8. 500 cells nextera tagmentation 9. 100 cells nextera tagmentation 10. 6 ng purified DNA nextera tagmentation 11. 600 pg purified DNA nextera tagmentation 12. 60 pg purified DNA nextera tagmentation 13. 1000 cells MEFs IVT 14. 1000 cells MEFs nextera tagmentation 15. 1000 cells lysed without transposome complex IVT 16. pure DNA only (6 ng) IVT 17. Nuclease free H20 only IVT 18. 1000 cells lysed without transposome complex nextera tagmentation 19. pure DNA only (6 ng) nextera tagmentation 20. Nuclease free H20 only nextera tagmentation
- 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 and 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
- incubate below mixtures at 37C for 30 mins.
' | 1. 1K IVT | 2. 500 IVT | 3. 100 IVT | 7. 1K nxta | 8. 500 nxta | 9. 100 nxta | 13. 1K MEF IVT | 14. 1K MEF nxta | 15. 1K IVT con | 17. NTC IVT | 18. 1K nxta con | 20. NTC nxta |
cells | 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 |
4. transposition reaction, using (T7tspn-top2)
- add the below into one tube and incubate for 8 minutes at 55C.
1 uL nextera HMW 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)
- IVT on filled in reaction and not filled in reaction in to show fill-in is working.
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
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. DNase I digestion and Zymo RNA clean and concentrator cleanup. (removes blocking primer and left over transposon too so wont be later possible PCR issues)
- Resuspend in appropriate volume in nuclease free H2O.
- not doing protease digestion since seems it wasnt as effective in previous RNase III fragmentation
11. Quanitate RNA with QUbit
12. 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)
13. 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
14. second strand synthesis (qPCR) (KAPA)
KAPA SYBR FAST qPCR mix X35 cycles 25 uL KAPA SYBR 4 uL primers, 1 uL F, 1 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX] (23,24)) 1 uL H2O 20 uL DNA template (use 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 before curves saturate (usually cycle 6-7)
15.qiaquick cleanup
- can quanitate with nanodrop
- run qiaquick to clean sample before performing qPCR.
16. Gel Size selection
- gel size select from 400-800 bp, follow gel size selection protocol
- do not need to include controls.
17. Cloning and Transformation, then genewiz sequencing for verification of inserts
18. Submit for sequencing if genewiz sequencing checks out.
Results
- bal hbal bah a
Notes ETC
- 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?