Brandon:LabNotes/Project1/2013-7-2: Difference between revisions

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13. second strand synthesis (qPCR) (KAPA), addition of barcodes
13. second strand synthesis (qPCR) (KAPA), addition of barcodes
  Samples:
  Samples:
Samples:
  Index 61, 1. RNase III, 1200 cells
  Index 65, N2 adaptor  1. RNase III, 1200 cells
  Index 62, 2. RNase III, 1200 cells
  Index 66, N2 adaptor  2. RNase III, 1200 cells
  Index 63, 3. RNase III, 1200 cells 1 hour freeze
  Index 67, N2 adaptor  3. RNase III, 1200 cells 1 hour freeze
  Index 64, 4. RNase III, 1200 cells 1 hour freeze
  Index 68, N2 adaptor  4. RNase III, 1200 cells 1 hour freeze
  Index 65, 5. RNase III, 3000 cells 1 hour freeze
  Index 69, N2 adaptor  5. RNase III, 3000 cells 1 hour freeze
  Index 66, 6. Rnase III, pure Jurkat DNA control
  Index 70, N2 adaptor  6. Rnase III, pure Jurkat DNA control
  Index 67, 7. RNase III, 1200 cells lysed without transposome complex IVT
  Index 71, N2 adaptor  7. RNase III, 1200 cells lysed without transposome complex IVT
  Index 68, 8. RNase III, Nuclease free H20 only IVT
  Index 72, N2 adaptor  8. RNase III, Nuclease free H20 only IVT
Index 69, 9. Rnase III, pure DNA, T7-top2-PCR-iaf2
Index 70, 10. Rnase III, pure DNA, T7-top2-PCR-iaf3
Index 71, 11-2. RNase III, 1200 cells, T7-top2-PCR-iaf2
Index 72, 12-2. Rnase III, 1200 cells, T7-top2-PCR-iaf3
   
   
  KAPA SYBR FAST qPCR mix until saturation, X35 cycles
  KAPA SYBR FAST qPCR mix until saturation, X35 cycles
   
   

Revision as of 00:24, 4 July 2013

IVT Round 8, Fresh versus Frozen cells redo2

  • USE PBS TO RESUSPEND CELLS!!! mistake made with Round 7


  • Round 5, frozen versus live cells gave all good results RNA generation after IVT on TBU gel, sequencing read statistics, and TBE gel on barcoded samples. however when performing coverageBed, samples did not have good overlap with UCSC GM12878 data. Thus repeating the assay, and also using beads purification since it has been optimizied.


  • for beads purification, RNA beads will be used. 30 uL of water will be added to the IVT sample, followed by 100 uL of RNA beads. Drying time after the washes will be 3 minutes. Elutions will be saved just in case samples did not bind to the beads.



Reasons why fresh cells are needed for DNase I accessibility assays

  • For DNase I accessibility assays fresh cells are always used. This is how it has been done for DNase I assays, which are mainly used for DNase accessibility. According to this article, Rapid genome-scale mapping of chromatin accessibility in tissue several reasons why you need fresh cells and some drawbacks of DNase I accessibility assays.
    • DNase I has narrow effective concentration window (thus need titrations etc)
    • inhibited by high levels of actin, so each sample must be individually titrated.
    • usually nuclei are isolated prior to DNase I digestion, since it reduced actin concentration.
  • since nuclei need to be obtained fresh cells have to be used.
  • Thus if the assay could be used on frozen cells, that would increase its ability to be used in a wider variety of contexts.


  • testing on 1000 cell samples since that gives the best data


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



Before starting protocols

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 this time:

Samples:
Index 65, N2 adaptor  1. RNase III, 1200 cells
Index 66, N2 adaptor  2. RNase III, 1200 cells
Index 67, N2 adaptor  3. RNase III, 1200 cells 1 hour freeze
Index 68, N2 adaptor  4. RNase III, 1200 cells 1 hour freeze
Index 69, N2 adaptor  5. RNase III, 3000 cells 1 hour freeze
Index 70, N2 adaptor  6. Rnase III, pure Jurkat DNA control
Index 71, N2 adaptor  7. RNase III, 1200 cells lysed without transposome complex IVT
Index 72, N2 adaptor  8. RNase III, Nuclease free H20 only IVT


IVT Protocol

  • 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


2.5 freeze cells

  • a. count cells, resuspend in PBS so much concentrated, or at the right concentration for experiments (1000 cells/ul, 5000 cells/ul)
  • b. put into EP DNA low bind tubes and snap freeze with liquid nitrogen. after snap freezing, transfer to -80C freezer.
  • c. freeze in -80C for 1 hour. Take out after 1 hour and let thaw ON ICE. Leave cells on ice and continue with downstream procedures.


3. Prepare samples, lyse cells with lysis buffer

Samples:
1. RNase III, 1200 cells
2. RNase III, 1200 cells
3. RNase III, 1200 cells 1 hour freeze
4. RNase III, 1200 cells 1 hour freeze
5. RNase III, 3000 cells 1 hour freeze
6. Rnase III, pure Jurkat DNA control
7. RNase III, 1200 cells lysed without transposome complex IVT
8. RNase III, 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.
7/2/2013 ' ' ' ' ' ' ' ' '
IVT Round 7, Fresh versus Frozen cells redo 5X lysis buffer for 3K cells
smpl_ID DNA lysis_buf LMW tspn H2O total protease total taq2x
1. RNase III, 1000 cells 1.344 1.344 1 0.8 0.512 5.000 1 6.000 6.000
2. RNase III, 1000 cells 1.344 1.344 1 0.8 0.512 5.000 1 6.000 6.000
3. RNase III, 1000 cells 1 hour freeze 1.344 1.344 1 0.8 0.512 5.000 1 6.000 6.000
4. RNase III, 1000 cells 1 hour freeze 1.344 1.344 1 0.8 0.512 5.000 1 6.000 6.000
5. RNase III, 3000 cells 1 hour freeze 3.361 0.84 1 0.8 - 6.001 1 7.001 6.000
6. Rnase III, pure Jurkat DNA control 1 1 1 0.8 1.200 5.000 1 6.000 6.000
7. RNase III, 1000 cells lysed without transposome complex IVT 1.344 1.344 1 0 1.312 5.000 1 6.000 6.000
8. RNase III, Nuclease free H20 only IVT 0 0 1 0 4.000 5.000 1 6.000 6.000
Samples:
1. RNase III, 1200 cells
2. RNase III, 1200 cells
3. RNase III, 1200 cells 1 hour freeze
4. RNase III, 1200 cells 1 hour freeze
5. RNase III, 3000 cells 1 hour freeze
6. Rnase III, pure Jurkat DNA control
7. RNase III, 1200 cells lysed without transposome complex IVT
8. RNase III, Nuclease free H20 only IVT

4. transposition reaction, using (T7tspn-top2)


  • samples 7-8 get H2O instead of transposome
  • 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 AMPURE RNA beads. elute in 10 uL or less.

  • Protocol changes:
    • 1. add 30 uL to IVT reaction to bring to 50 uL total before starting protocol.
    • 2. Use 1.8:1 ratio of beads, thus 80 uL of beads for 50 uL of sample in this case
    • 3. Drying time after the washes is 3 minutes.
  • 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, 1. RNase III, 1200 cells
Index 62, 2. RNase III, 1200 cells
Index 63, 3. RNase III, 1200 cells 1 hour freeze
Index 64, 4. RNase III, 1200 cells 1 hour freeze
Index 65, 5. RNase III, 3000 cells 1 hour freeze
Index 66, 6. Rnase III, pure Jurkat DNA control
Index 67, 7. RNase III, 1200 cells lysed without transposome complex IVT
Index 68, 8. RNase III, Nuclease free H20 only IVT
Index 69, 9. Rnase III, pure DNA, T7-top2-PCR-iaf2
Index 70, 10. Rnase III, pure DNA, T7-top2-PCR-iaf3
Index 71, 11-2. RNase III, 1200 cells, T7-top2-PCR-iaf2
Index 72, 12-2. Rnase III, 1200 cells, T7-top2-PCR-iaf3


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

  • RNA quantification results


  • RNA after ~16.5 hours IVT, run on TBU gel



future and conclusions

  • blah

Blah