Brandon:LabNotes/Project1/2012-8-6: Difference between revisions

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10. DNase I digestion and Zymo RNA clean and concentrator cleanup. removes dsDNA. does not remove ss or DNA:RNA hybrids at high efficiency, 1:500 that of dsDNA. better than nothing...
10. (ONLY DID ZYMO CLEANUP, STILL WORKED FINE 2012/08/28 update) DNase I digestion and Zymo RNA clean and concentrator cleanup. removes dsDNA. does not remove ss or DNA:RNA hybrids at high efficiency, 1:500 that of dsDNA. better than nothing...
*Resuspend in appropriate volume in nuclease free H2O.
*Resuspend in appropriate volume in nuclease free H2O.
*not doing protease digestion since seems it wasnt as effective in previous [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-6-6 RNase III fragmentation]
*not doing protease digestion since seems it wasnt as effective in previous [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-6-6 RNase III fragmentation]
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18. Submit for sequencing if genewiz sequencing checks out.
18. Submit for sequencing if genewiz sequencing checks out.


===Results===
===Results===
Line 592: Line 590:
   
   
[[File:ZhangLab 2 2012-08-17 15hr 12min-labeled.jpg|600px]]
[[File:ZhangLab 2 2012-08-17 15hr 12min-labeled.jpg|600px]]
'''gel size selection'''
*after gel size selection for IVT samples and for nextera samples
*quanitated DNA in smears on gels from above and combined IVT samples, and nextera samples separately since are barcoded already.
*used same amount of DNA per sample except samples that did not have enough.
*IVT samples
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
| ||for 100 ng total each sample||
|-
| 1000 cell||2.428363987||uL
|-
| 500 cell||3.389656805||uL
|-
| 100 cell||8.85869023||uL
|-
| 6 ng||2.587997939||uL
|-
| 600 pg||11.27038606||uL
|-
| 60 pg||50.15563592||use 20 uL of sample.
|-
| 1000 MEF||2.748981532||uL
|-
| ||||
|-
| ||||~700 ish ng total
|-
| ||||2 wells on gel
|-
| ||51.43971247||
|-
|
|}
*nextera samples
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''sample'''
| align="center" style="background:#f0f0f0;"|'''ng'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
| 1000 cell||5.882608179||uL||
|-
| 500 cell||2.655553673||uL||
|-
| 100 cell||1.896579441||uL||
|-
| 6 ng||8.937349074||uL||
|-
| 600 pg||-567.899155||9||uL
|-
| 60 pg||-197.013903||9||uL
|-
| 1000 MEF||3.599657838||uL||
|-
| ||||||
|-
| ||||||
|-
| ||40.97174821||500 ng total, 2 wells||
|-
|
|}
*gel size selection gel check
[[File:ZhangLab 2 2012-08-21 16hr 38min-labeled.jpg|600px]]
'''cloning and transformation with gel size selected sample'''
*alot did not amplify, not sure why. though if one worked then it is still comparable to the cloning/transformation that was done [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-6-29#update_7.2F6.2F2012_IVT_on_pure_DNA_samples.2FIVT_again_on_cells with the random nonamer]
**gave normal ratios of blue to white colonies
**could be that need to do transformation again.
**PCR didn't work? could redilute primers, use diff MM etc.
**ends not A-tailed well enough?
**POSITIVE result is that uninformative fragments (330 bp) are not present. not sure why though.
*send in samples 1,3,4,8,12,13,14,16,24,33,42,43 for sequencing
*IVT with RNase III fragmentation has better cloning/transformation statistics than [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-6-29 random nonamer method], which out of 77 clones, 10.3% had a valid genomic insert. half of them had incorrect ends, thus true statistics were around 5%. all in this analysis had correct 5', 3' ends for sequencing.
*sequence analysis file [[Media:2012-08-24 Rnase III fragmentation protocol.docx]]
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''samples'''
| align="center" style="background:#f0f0f0;"|'''possible'''
| align="center" style="background:#f0f0f0;"|'''validated'''
| align="center" style="background:#f0f0f0;"|'''% val'''
|-
| IVT||1-22||8||4||.1818
|-
| nextera||23-44||4||3||.13536
|-
|
|}
[[File:ZhangLab 2 2012-08-23 14hr 09min-labeled.jpg|600px]]
[[File:ZhangLab 2 2012-08-23 14hr 11min-labeled.jpg|600px]]
[[File:ZhangLab 2 2012-08-23 14hr 19min-labeled.jpg|600px]]
[[File:ZhangLab 2 2012-08-23 14hr 20min-labeled.jpg|600px]]


===Notes ETC===
===Notes ETC===
Line 768: Line 883:




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
Stuff used for RNA-seq for BENG160 class

Latest revision as of 01:06, 29 August 2012

Custom transposon with IVT amplification, round 2[edit]

  • 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[edit]

  • 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[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

  • nextera transposomes
  • transposase/transposome
  • IVT reaction mixture
  • cells etc
  • blocking primer, RNase III, PolyA Polymerase


2. Purify GM12878 DNA from Gm12878 cells with DNeasy blood and tissue kit for cells (uses proteinase). quanititate DNA and aliquot to a standard concentration of 60 ng or a multiple of that.

3. Have to raise off MEFs with trypsin

1. suck off media
2. add 5 mL PBS/wash
3. suck off
4. 2 mL trypsin
5. put in 37C, 2 minutes
6. dilute with PBS, 10 mL
7. spin down and resuspend.

4. samples

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

Nextera Tagmentation Protocol[edit]

1. See step 3 in IVT protocol. Do the cell lysis section for the "nextera tagmentation samples", samples 7,8,9,14,18.


2. Perform tagmentation on lysed cells and pure DNA. with 1:10 diluted nextera enzyme. (1:50 diluted in reaction)

Dilute the enzyme: 1:10     
For each rxn, used mix of: 
 1ul 5x LMW Buffer
 2ul cell lysate
 1ul diluted enzyme
 1ul H2O
----------------------------
5ul total / reaction
55C 10 min


3. Protease 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, Nxtra atpr  7.  1000 cells nextera tagmentation
Index 56, Nxtra atpr  8.  500  cells nextera tagmentation
Index 57, Nxtra atpr  9.  100  cells nextera tagmentation
Index 58, Nxtra atpr  10. 6   ng purified DNA nextera tagmentation
Index 59, Nxtra atpr  11. 600 pg purified DNA nextera tagmentation
Index 60, Nxtra atpr  12. 60  pg purified DNA nextera tagmentation
Index 62, Nxtra atpr  14. 1000 cells MEFs nextera tagmentation
Index 66, Nxtra atpr  18. 1000 cells lysed without transposome complex nextera tagmentation
Index 67, Nxtra atpr  19. pure DNA only (6 ng) nextera tagmentation
Index 68, Nxtra atpr  20. 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 5 -> 72C 3min.

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.  600 pg purified DNA IVT
6.  60  pg purified DNA IVT
13. 1000 cells MEFs IVT
15. 1000 cells lysed without transposome complex IVT
16. pure DNA only (6 ng) IVT
17. 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 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 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


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. (ONLY DID ZYMO CLEANUP, STILL WORKED FINE 2012/08/28 update) DNase I digestion and Zymo RNA clean and concentrator cleanup. removes dsDNA. does not remove ss or DNA:RNA hybrids at high efficiency, 1:500 that of dsDNA. better than nothing...

  • 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)

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), 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.  600 pg purified DNA IVT
Index 54, N2 adaptor  6.  60  pg purified DNA IVT
Index 61, N2 adaptor  13. 1000 cells MEFs IVT
Index 63, N2 adaptor  15. 1000 cells lysed without transposome complex IVT
Index 64, N2 adaptor  16. pure DNA only (6 ng) IVT
Index 65, N2 adaptor  17. Nuclease free H20 only IVT

KAPA SYBR FAST qPCR mix until saturation, X35 cycles

25 uL KAPA SYBR
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)


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[edit]

Nextera results and gels

  • nanodrop results for nextera samples
Sample ID smpl # ng/ul
1000 cell 7 75.36
500 cell 8 88.23
100 cell 9 55.61
6 ng 10 61.54
600 pg 11 9.69
60 pg 12 3.99
MEF 14 107.29
NTC 18 4.44
NTC 19 4.82
NTC 20 9.31



  • ran gel after 2 step AMPure beads purification (addition of illuminia primers, barcodes)
    • Interestingly more sample is detected with lower cell concentrations. Probably because with lower cell concentrations there are more viable fragments created, since more insertions will happen over less DNA. Same amount of transposome was used for each reaction. Nonviable fragments could be created by wrong ends and distance between insertions is to far.
    • Not enough DNA for samples 11, 12?
    • MEF's (14) and NTCs worked as expected.

File:ZhangLab 2 2012-08-16 12hr 38min-labeled.jpg



  • qPCR curves for blue/orange amplification of products
1. 1000 cells nextera
2. 500 cells nextera
3. 100 cells nextera
4. 6 ng purified DNA nextera
5. 600 pg purified DNA nextera
6. 60 pg purified DNA nextera
7. 1000 cells MEFs nextera
8. 1000 cells lysed without transposome nextera
9. pure DNA only nextera
10. N-H2O only nextera

600[x



IVT amplification results/gels

  • Used 1 uL for TBU gel, 2 uL for Qubit quantitation
' [Sample] ng/uL eluted (uL) total RNA ng
1000 cell 43.3 ng/uL 14 606.2 ng
500 cell 29.3 ng/uL 14 410.2 ng
100 cell 15.3 ng/uL 14 214.2 ng
6 ng 43.1 ng/uL 14 603.4 ng
600 pg 6.4 ng/uL 14 89.6 ng
60 pg Out of Range
MEF 59 ng/uL 14 826 ng
NTC Out of Range
NTC Out of Range
NTC Out of Range


  • graph of ng RNA versus cell number for IVT RNA amplification. Linear based on cell number.

File:Ng RNA vs cell number IVT.png


  • RNA after IVT amplification, TBU gel

File:ZhangLab 2 2012-08-16 16hr 05min-labeled.jpg



  • qPCR curves for barcode/adaptor addition
  • Negative controls amplified thus wierd, but looks fine when running TBE gel.
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 MEFs IVT
8. 1000 cells lysed without transposome IVT
9. pure DNA only IVT
10. N-H2O only IVT

File:2012-08-15 IVT accessibility barcode addition.bmp



  • 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 2012-08-17 15hr 12min-labeled.jpg



gel size selection

  • after gel size selection for IVT samples and for nextera samples
  • quanitated DNA in smears on gels from above and combined IVT samples, and nextera samples separately since are barcoded already.
  • used same amount of DNA per sample except samples that did not have enough.
  • IVT samples
' ' '
for 100 ng total each sample
1000 cell 2.428363987 uL
500 cell 3.389656805 uL
100 cell 8.85869023 uL
6 ng 2.587997939 uL
600 pg 11.27038606 uL
60 pg 50.15563592 use 20 uL of sample.
1000 MEF 2.748981532 uL
~700 ish ng total
2 wells on gel
51.43971247


  • nextera samples
sample ng ' '
1000 cell 5.882608179 uL
500 cell 2.655553673 uL
100 cell 1.896579441 uL
6 ng 8.937349074 uL
600 pg -567.899155 9 uL
60 pg -197.013903 9 uL
1000 MEF 3.599657838 uL
40.97174821 500 ng total, 2 wells


  • gel size selection gel check

File:ZhangLab 2 2012-08-21 16hr 38min-labeled.jpg


cloning and transformation with gel size selected sample

  • alot did not amplify, not sure why. though if one worked then it is still comparable to the cloning/transformation that was done with the random nonamer
    • gave normal ratios of blue to white colonies
    • could be that need to do transformation again.
    • PCR didn't work? could redilute primers, use diff MM etc.
    • ends not A-tailed well enough?
    • POSITIVE result is that uninformative fragments (330 bp) are not present. not sure why though.
  • send in samples 1,3,4,8,12,13,14,16,24,33,42,43 for sequencing


' samples possible validated % val
IVT 1-22 8 4 .1818
nextera 23-44 4 3 .13536


File:ZhangLab 2 2012-08-23 14hr 09min-labeled.jpg File:ZhangLab 2 2012-08-23 14hr 11min-labeled.jpg File:ZhangLab 2 2012-08-23 14hr 19min-labeled.jpg File:ZhangLab 2 2012-08-23 14hr 20min-labeled.jpg




Notes ETC[edit]

  • since previous frag block was designed for T7-top3.
T7-frag-block-top2
5'- CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGATCTCCC/3InvdT/  Tm= 81.45



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.  600 pg purified DNA IVT
Index 54, N2 adaptor  6.  60  pg purified DNA IVT
Index 55, Nxtra atpr  7.  1000 cells nextera tagmentation
Index 56, Nxtra atpr  8.  500  cells nextera tagmentation
Index 57, Nxtra atpr  9.  100  cells nextera tagmentation
Index 58, Nxtra atpr  10. 6   ng purified DNA nextera tagmentation
Index 59, Nxtra atpr  11. 600 pg purified DNA nextera tagmentation
Index 60, Nxtra atpr  12. 60  pg purified DNA nextera tagmentation
Index 61, N2 adaptor  13. 1000 cells MEFs IVT
Index 62, Nxtra atpr  14. 1000 cells MEFs nextera tagmentation
Index 63, N2 adaptor  15. 1000 cells lysed without transposome complex IVT
Index 64, N2 adaptor  16. pure DNA only (6 ng) IVT
Index 65, N2 adaptor  17. Nuclease free H20 only IVT
Index 66, Nxtra atpr  18. 1000 cells lysed without transposome complex nextera tagmentation
Index 67, Nxtra atpr  19. pure DNA only (6 ng) nextera tagmentation
Index 68, Nxtra atpr  20. 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