Brandon:LabNotes/Project1/2013-2-28
IVT round 6, Tet1 WT/KO's in somatic and PGC cells[edit]
- IVT round 5 showed that cells frozen in -80 worked just as good as fresh cells from culture. seqeuencing analysis has not been done, but all other metrics of IVT RNA amounts, qPCR barcode addition and gels have shown it to work. have submitted for sequencing.
- for rare cells, they have been frozen in -80. and thus same procedure will be performed. Only issue is varying volumes which will be dealt with. probably centrifuge samples X300g and resuspend in limited volume..
- Since IVT R4 and Rui's recent RNase III experiment have shown RNase III to be better, will use only RNase III.
- testing on 1000 cell samples. Will also do 10000 as a safety measure.
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
- RNA clean and concentrator
- IVT reaction mixture
- cells etc
- blocking primer, RNase III, PolyA Polymerase, ATP, taq polymerase, other primers
2. samples this time:
- description: all from male mice. E13.5 PGC samples from GW Na-Bi sequencing originally. KO's are for Tet1. cells were sorted by FACS into respective tubes. about 6 months old (from September 2012)
smpl_ID | smpl_rep | sex | geno | cell_type | cell_num | ' | volume |
1- | 1 | male | WT | somatic | 62,459 | 2x 10K | ~100 uL |
3- | 2 | male | WT | somatic | 27,813 | 2x 1K | ~70 uL |
1+ | 1 | male | WT | PGC | 8,213 | 2x 1K | ~10 uL |
3+ | 2 | male | WT | PGC | 2,495 | --- | ~20 uL |
5- | 1 | male | -/- | somatic | 34,924 | 2x10K | ~100 uL |
8- | 2 | male | -/- | somatic | 32,766 | 2x1K | ~100 uL |
5+ | 1 | male | -/- | PGC | 5,888 | 2x1K | ~20 uL |
8+ | 2 | male | -/- | PGC | 1,876 | --- | ~8 uL |
Samples: Index 61, N2 adptr, 1. 1- WT somatic 10K cells rep1 Index 62, N2 adptr, 2. 1- WT somatic 10K cells rep2 Index 63, N2 adptr, 3. 3- WT somatic 1K cells rep1 Index 64, N2 adptr, 4. 3- WT somatic 1K cells rep2 Index 65, N2 adptr, 5. 3+ WT PGC 1K cells rep1 (pretty sure used 1+) Index 66, N2 adptr, 6. 3+ WT PGC 1K cells rep2 (pretty sure used 1+) Index 67, N2 adptr, 7. 5- KO somatic 10K cells rep1 Index 68, N2 adptr, 8. 5- KO somatic 10K cells rep2 Index 69, N2 adptr, 9. 8- KO somatic 1K cells rep1 Index 70, N2 adptr, 10. 8- KO somatic 1K cells rep2 Index 71, N2 adptr, 11. 5+ KO PGC 1K cells rep1 Index 72, N2 adptr, 12. 5+ KO PGC 1K cells rep2 Index 60, N2 adptr, 13. NTC
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
Samples: 1. 1- WT somatic 10K cells rep1 2. 1- WT somatic 10K cells rep2 3. 3- WT somatic 1K cells rep1 4. 3- WT somatic 1K cells rep2 5. 3+ WT PGC 1K cells rep1 (pretty sure used 1+) 6. 3+ WT PGC 1K cells rep2 (pretty sure used 1+) 7. 5- KO somatic 10K cells rep1 8. 5- KO somatic 10K cells rep2 9. 8- KO somatic 1K cells rep1 10. 8- KO somatic 1K cells rep2 11. 5+ KO PGC 1K cells rep1 12. 5+ KO PGC 1K cells rep2 13. NTC
- 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.
- 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 with different concentrations 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. Ensure solution concentrations are at 1X of lysis buffer. aim for desired cell number in 1.6 uL (use 2X lysis buffer) cell buffer, or 4 uL (need 1 uL 5X lysis buffer then) at most.
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. elute in 10 uL or less.
- can quantitate with Qubit or on TBU gel.
9. RNase III fragmentation (NEB), Mg++ fragmentation:
Mg++ fragmentation:
a. RNA fragmentation with MgCl and end repair with T4PNK X uL RNA (100ng-200 ng) .5 uL 10x Fragmentation buffer (or dilute to 5X and use 1 uL) 3-X uL Nuclease-free H2O __________ 5 uL Total b. Incubate at 94C for 5 min, place the tube on ice. Then perform the end repair as following: 5 uL RNA (100 ng total) .8 uL 10x Polymerase buffer (PAP buffer) .8 uL 10mM ATP 1.4 uL T4PNK enzyme ____________ 8 uL Total 37 C for 30 mins. c. proceed to PAP addition when ready.
RNase III fragmentation:
use 100-200 ng of RNA 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 RT for 5 minutes (mix well) 2. Mix the following components in a sterile PCR tube: a. 5 uL Purified mRNA + blocking primer (50-250 nanograms) .5 uL RNase III (1 unit/μl) .8 uL RNase III Reaction Buffer (10X) X uL Nuclease-Free Water add in DNA primer to protect 5' end since don't want degradation?? ____________ 8.5 uL total volume b. Incubate in a preheated thermal cycler for 5 minutes at 37°C. c. Heat inactivate RNase III at 65C for 20 minutes. d. Immediately cool on ice and proceed to PAP addition.
10. Poly(A) Addition with polyA polymerase (Enzymatics)
- enzymatics PolyA polymerase.
ALL RNA SAMPLES
a. to each each add: 1.2 uL polyA enzyme .8 uL 10 mM ATP ________ 10.5 uL (should be) b. Incubate at 37C for 10 minutes c. proceed immediately to Zymo cleaning.
11. Zymo RNA clean and concentrator cleanup.
- Resuspend in appropriate volume in nuclease free H2O (10 uL last time)
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 for 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 61, N2 adptr, 1. 1- WT somatic 10K cells rep1 Index 62, N2 adptr, 2. 1- WT somatic 10K cells rep2 Index 63, N2 adptr, 3. 3- WT somatic 1K cells rep1 Index 64, N2 adptr, 4. 3- WT somatic 1K cells rep2 Index 65, N2 adptr, 5. 3+ WT PGC 1K cells rep1 (pretty sure used 1+) Index 66, N2 adptr, 6. 3+ WT PGC 1K cells rep2 (pretty sure used 1+) Index 67, N2 adptr, 7. 5- KO somatic 10K cells rep1 Index 68, N2 adptr, 8. 5- KO somatic 10K cells rep2 Index 69, N2 adptr, 9. 8- KO somatic 1K cells rep1 Index 70, N2 adptr, 10. 8- KO somatic 1K cells rep2 Index 71, N2 adptr, 11. 5+ KO PGC 1K cells rep1 Index 72, N2 adptr, 12. 5+ KO PGC 1K cells rep2 Index 60, N2 adptr, 13. NTC
KAPA SYBR FAST qPCR mix until saturation, X35 cycles 12.5 uL KAPA SYBR FAST qPCR mix 2 uL primers, 2 uL F, 2 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX]) .5 uL H2O 10 uL DNA template (use half RT reaction) _____________ 25 uL 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]
- RNA quantification results
- amounts used for RNA fragmentation for 200 ng total of RNA for each sample.
update 3/22/2013
- (pretty sure used 1+) for 3+ samples
- end update
USED | ' | ' | ' | ' | ' | ' |
ng/ul | ttl_ng_in_10_uL | for_200_ng | 100_ng | H2O | ||
1. | 69.33154157 | 693.3154157 | 2.884689933 | 2.315310067 | 1. 1- WT somatic 10K cells rep1 | |
2. | 76.96649938 | 769.6649938 | 2.598533149 | 2.601466851 | 2. 1- WT somatic 10K cells rep2 | |
3. | 128.6813269 | 1286.813269 | 1.554227057 | 3.645772943 | 3. 3- WT somatic 1K cells rep1 | |
4. | 42.13522543 | 421.3522543 | 4.746622285 | 0.453377715 | 4. 3- WT somatic 1K cells rep2 | |
5. | 77.70853716 | 777.0853716 | 2.573719791 | 2.626280209 | 5. 3+ WT PGC 1K cells rep1 | |
6. | 21.07504403 | 210.7504403 | 4.7449 | 0.4551 | 6. 3+ WT PGC 1K cells rep2 | |
7. | 201.4960462 | 2014.960462 | 0.992575307 | 4.207424693 | 7. 5- KO somatic 10K cells rep1 | |
8. | 155.3745007 | 1553.745007 | 1.287212503 | 3.912787497 | 8. 5- KO somatic 10K cells rep2 | |
9. | 55.19250842 | 551.9250842 | 3.623680201 | 1.576319799 | 9. 8- KO somatic 1K cells rep1 | |
10. | 30.88817103 | 308.8817103 | 3.237485311 | 1.962514689 | 10. 8- KO somatic 1K cells rep2 | |
11. | 48.54510953 | 485.4510953 | 4.119879468 | 1.080120532 | 11. 5+ KO PGC 1K cells rep1 | |
12. | 47.96293442 | 479.6293442 | 4.169886651 | 1.030113349 | 12. 5+ KO PGC 1K cells rep2 | |
- RNA after ~16.5 hours IVT, run on TBU gel
- isn't consistent between samples, probably differing amounts of cells in each one.
File:ZhangLab 2 2013-03-01 14hr 17min-labeled.jpg File:ZhangLab 2 2013-03-01 14hr 21min-labeled.jpg
- barcode addition PCR curves. terminated at end of 5th cycle since thats what did when run before.
- !!!!USED TAQ2X INSTEAD OF KAPA SYBR FAST, THUS CURVES WOULD NOT GO UP. HAVE 10 UL CDNA LEFT IF NEED TO REPEAT!!!!
update 3/22/2013
- (pretty sure used 1+) for 3+ samples
- end update
- Curves are in order of sample listings below.
Samples: Index 61, N2 adptr, 1. 1- WT somatic 10K cells rep1 Index 62, N2 adptr, 2. 1- WT somatic 10K cells rep2 Index 63, N2 adptr, 3. 3- WT somatic 1K cells rep1 Index 64, N2 adptr, 4. 3- WT somatic 1K cells rep2 Index 65, N2 adptr, 5. 3+ WT PGC 1K cells rep1 Index 66, N2 adptr, 6. 3+ WT PGC 1K cells rep2 Index 67, N2 adptr, 7. 5- KO somatic 10K cells rep1 Index 68, N2 adptr, 8. 5- KO somatic 10K cells rep2 Index 69, N2 adptr, 9. 8- KO somatic 1K cells rep1 Index 70, N2 adptr, 10. 8- KO somatic 1K cells rep2 Index 71, N2 adptr, 11. 5+ KO PGC 1K cells rep1 Index 72, N2 adptr, 12. 5+ KO PGC 1K cells rep2 Index 60, N2 adptr, 13. NTC
File:2013-03-01 round 6 pgc and somatic cells, rnase iii.bmp
- TBE gels after barcode addition
File:ZhangLab 2 2013-03-04 10hr 56min-labeled.jpg File:ZhangLab 2 2013-03-04 10hr 58min-labeled.jpg
- input amounts for gel size selection
update 3/22/2013
- (pretty sure used 1+) for 3+ samples
- end update
' | ng/ul | for_65_ng | sample | ' | ' |
1. | 15.92390661 | 4.081912912 | Index 61, N2 adptr, 1. 1- WT somatic 10K cells rep1 | ||
2. | 18.50057412 | 3.513404481 | Index 62, N2 adptr, 2. 1- WT somatic 10K cells rep2 | ||
3. | 11.86041029 | 5.480417492 | Index 63, N2 adptr, 3. 3- WT somatic 1K cells rep1 | ||
4. | 22.33986829 | 2.909596384 | Index 64, N2 adptr, 4. 3- WT somatic 1K cells rep2 | ||
5. | 13.79665946 | 4.71128538 | Index 65, N2 adptr, 5. 3+ WT PGC 1K cells rep1 | ||
6. | 10.98216681 | 5.918686276 | Index 66, N2 adptr, 6. 3+ WT PGC 1K cells rep2 | ||
7. | 9.102086556 | 7.141219719 | Index 67, N2 adptr, 7. 5- KO somatic 10K cells rep1 | ||
8. | 9.730105117 | 6.68029782 | Index 68, N2 adptr, 8. 5- KO somatic 10K cells rep2 | ||
9. | 16.58928621 | 3.918191486 | Index 69, N2 adptr, 9. 8- KO somatic 1K cells rep1 | ||
10. | 13.36647449 | 4.862912808 | Index 70, N2 adptr, 10. 8- KO somatic 1K cells rep2 | ||
11. | 21.40411288 | 3.036799534 | Index 71, N2 adptr, 11. 5+ KO PGC 1K cells rep1 | ||
12. | 23.34285554 | 2.784577914 | Index 72, N2 adptr, 12. 5+ KO PGC 1K cells rep2 | ||
55.03930221 | 65 | ng/sample | |||
780 | ng ttl for 200-1000 bp | ||||
- gel size selection
update 3/22/2013
- (pretty sure used 1+) for 3+ samples
- end update
File:ZhangLab 2 2013-03-04 15hr 02min.jpg
- validation of gel size selection:
- is light, might have to do again and concentrate sample
File:ZhangLab 2 2013-03-04 18hr 09min-labeled.jpg
- gel size selection, second time.
update 3/22/2013
- (pretty sure used 1+) for 3+ samples
- end update
File:ZhangLab 2 2013-03-05 11hr 10min.jpg
- first gel size selection concentrated with concentrator from 20 uL to 5 uL
- second sample is fresh gel size selection
- in end, second sample concentrated to ~7 uL and then combined with first sample and submitted for sequencing.
File:ZhangLab 2 2013-03-05 15hr 10min-labeled.jpg
future and conclusions[edit]
update 3/22/2013
- (pretty sure used 1+) for 3+ samples
- end update
- blah balh
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