Brandon:LabNotes/Project1/2013-2-28

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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)

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

Blah