Brandon:LabNotes/Project1/2013-8-19

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New RNA processing method, No PAP, No RnIII on cells/FACS buffer for accessibility testing on R10 samples


  • Had very high single read alignments (76-79% with pure DNA, and 200 ng of RNA was used for each sample. Clonal reads and unmapped reads were low. At best see 60% single read alignments for RnIII/PAP method for pure DNA, thus looking really promising.



  • can also adding in Rnase H, NPA primer and polymerase into the reaction at once. However since extension will be wanted off of the NPA primer, denaturing will probably still be needed.



Before Starting Protocol

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • T7-top2 transposomes
  • custom nextera transposomes
  • transposase/transposome
  • IVT reaction mixture
  • cells etc
  • Rnase H, random hexamers, protease, AMPURE beads ,ATP, taq polymerase, other polymerases (BST, klewnow exo-, DNAPI), other primers


  • 2. samples this time, 200 ng each sample if not stated.
Samples:
Index 60, NXTA adaptor  1. PBS 1K cells
Index 61, NXTA adaptor  2. PBS 12 uL 1000 cells
Index 62, NXTA adaptor  3. FACS buffer 1K cells
Index 63, NXTA adaptor  4. FACS buffer 1K cells (FORGOT TO ADD: 5 mM MgCl2)
Index 64, NXTA adaptor  5. FACS buffer 12 uL 1000 cells
Index 65, NXTA adaptor  6. 6 ng pure Jurkat DNA
Index 66, NXTA adaptor  7. 1.- PBS 1K cells, Zymo DNA purified
Index 67, NXTA adaptor  8. 3.- FACS bf 1K cells Zymo DNA purified
Index 68, NXTA adaptor  9. 4.- FACS bf 1K cells 800 ng RNA 
Index 69, NXTA adaptor  10. 4.- FACS bf 1K cells 400 ng RNA 
Index 70, NXTA adaptor  11. 4.- FACS bf 1K cells 100 ng RNA
Index 71, NXTA adaptor  12. 4.- FACS bf 1K cells 50  ng RNA
Index 72, NXTA adaptor  13. 1000 cells lysed w/out tspn



Protocol

  • If need to make more transposome, do first 2 steps. If not goto step 3.

Generation of T7tspn-top2 transposome

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


Generation of custom barcode (3' side of fragments) nextera transposome

1. annealing of ME sequence to Nextera transposon sequence

    • a. Make 100 uM stock solution of (NXTA_ME_BOT, "pMENTS") and (NXTA_ME_B, "B-METS").
    • 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 Nextera 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:


  • 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. Aim for X number of cells (or pure genomic DNA) in 1 uL so that 1 uL of 2X lysis buffer can be used in the reaction.
  • Prepare 2X LB from 10X buffer. mineral oil optional.
  • if needed make 4 or 6 uL solutions. ensure that lysis buffer is 1X in as the final concentration.


  • incubate the mixtures at 37C for 30 mins.


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)



AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH AMPURE BEADS


Samples this time:

Samples:
Index 60, NXTA adaptor  1. PBS 1K cells
Index 61, NXTA adaptor  2. PBS 12 uL 1000 cells
Index 62, NXTA adaptor  3. FACS buffer 1K cells
Index 63, NXTA adaptor  4. FACS buffer 1K cells (FORGOT TO ADD: 5 mM MgCl2)
Index 64, NXTA adaptor  5. FACS buffer 12 uL 1000 cells
Index 65, NXTA adaptor  6. 6 ng pure Jurkat DNA
Index 66, NXTA adaptor  7. 1.- PBS 1K cells, Zymo DNA purified
Index 67, NXTA adaptor  8. 3.- FACS bf 1K cells Zymo DNA purified
Index 68, NXTA adaptor  9. 4.- FACS bf 1K cells 800 ng RNA 
Index 69, NXTA adaptor  10. 4.- FACS bf 1K cells 400 ng RNA 
Index 70, NXTA adaptor  11. 4.- FACS bf 1K cells 100 ng RNA
Index 71, NXTA adaptor  12. 4.- FACS bf 1K cells 50  ng RNA
Index 72, NXTA adaptor  13. 1000 cells lysed w/out tspn


  • Amounts of RNA and water to add for MMLV RT
8/19/2013 800 ng H20 800 200 ng H2O 200 100 ng H20 100 50 ng H20 50
Index 61, N2 adaptor 1. PBS 1K cells 0.7460 9.2540 0.3730 9.6270 0.1865 9.8135
Index 62, N2 adaptor 2. PBS 12 uL 1000 cells 1.0600 8.9400 0.5300 9.4700 0.2650 9.7350
Index 63, N2 adaptor 3. FACS buffer 1K cells 0.7152 9.2848 0.3576 9.6424 0.1788 9.8212
Index 64, N2 adaptor 4. FACS buffer 1K cells 2.117 7.883 0.5293 9.4707 0.2647 9.7353 0.1323 9.8677
Index 65, N2 adaptor 5. FACS buffer 12 uL 1000 cells 1.5562 8.4438 0.7781 9.2219 0.3890 9.6110
Index 66, N2 adaptor 6. 6 ng pure Jurkat DNA 0.4148 9.5852 0.2074 9.7926 0.1037 9.8963


8. single strand synthesis MMLV RT (Clontech)

20 uL reaction

1. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 12.5 uL with
   Nuclease free H2O

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 mM DTT
   1  uL N-H20
  .5 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  ____
   20 uL total

4. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes.

5. Terminate the reaction by heating at 70C for 10 minutes



9. RNase H digestion

  • Use .5 Units for 20 uL reaction. Stock is at 5 Units/ul, thus dilute 10X and use 1 uL per reaction.

a. Add 1 uL of 10X diluted Rnase H to the reaction.

b. Incubate for 20 minutes at 37C.


10. Second strand synthesis: Adding sss_NPA_prmr primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_NPA_prmr has a high Tm.

a. Add 2.5 uL of sss_NPA_prmr to each reaction. 

b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice.

c. Add 2 uL of dNTPs to each reaction.

d. Add polymerases etc to reactions

          DNAP1: Dilute 2X to 5U/uL and add 1 uL to the reaction. Incubate at 37C for 30 minutes
                 (stock conc. 10U/uL)

    Klenow exo-: Add 1 uL of 5U/uL. Incubate at 37C for 30 minutes.

        BST 2.0: Add 2.5 uL 10X BST buffer, dilute enzyme 2X to 4 U/ul, add 1 uL to the reaction.
                 Incubate at 65C for 30 minutes

 BST 2.0 No buf: dilute enzyme 2X to 4 U/ul, add 1 uL to the reaction. Incubate at 65C for 30 minutes

          taq2X: Add 25 uL, Incubate at 72C for 10 minutes
          
            NTC: Just add 1 uL of Klenow exo-


11. Clean DNA with with Zymo DNA Clean & Concentrator-5 Kit OR AMPURE beads depending. elute in 6 uL or less. (can also use qiaquick)

SAMPLES 7,8 CLEAN WITH ZYMO

REST OF SAMPLES CLEAN WITH AMPURE BEADS

USED AMPURE DNA BEADS AT 1:1.8X RATIO

can quantitate with Qubit or on TBU gel.



11.5. Eluted DNA into 20 uL per sample, since when eluting in smaller amounts more sample is lost. Thus after elution used vaccum centrifuge commentator to concentrate sample size to ~4-5 uL per sample.

  • Also ~4-5 uL per sample is preferred for transposition, which has always been performed in low volume solutions. Additionally less transposome will be needed when doing that.



12. Fragmenting and 3' End tagging with Custom Nextera transposome

1 uL nextera LMW buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
X uL Nuclase free H2O
.8 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL) 
___________
5 uL total solution


  • Incubate at 55C for 10 minutes, cool briefly on ice after



13. 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



14. Fill in reaction

  • Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)


CAN TRY JUST FINISHING UP WITH TAQ2X (DID THAT THIS TIME, ADDED 12.5 UL OF 1X TAQ2X TO SOLUTION AND STOPPED AT END OF 6TH CYCLE. ADDED SYBR GREEN .075X FINAL CONCENTRATION IN SOLUTION

15. Clean DNA with with Zymo DNA Clean & Concentrator-5 Kit OR AMPURE beads depending. elute in 10 uL or less. (can also use qiaquick) can quantitate with Qubit or on TBU gel.


16. PCR addition of barcodes MAKE SURE TO USE NEXTERA INDEXES!!!!!

Samples:
Index 60, NXTA adaptor  1. PBS 1K cells
Index 61, NXTA adaptor  2. PBS 12 uL 1000 cells
Index 62, NXTA adaptor  3. FACS buffer 1K cells
Index 63, NXTA adaptor  4. FACS buffer 1K cells (FORGOT TO ADD: 5 mM MgCl2)
Index 64, NXTA adaptor  5. FACS buffer 12 uL 1000 cells
Index 65, NXTA adaptor  6. 6 ng pure Jurkat DNA
Index 66, NXTA adaptor  7. 1.- PBS 1K cells, Zymo DNA purified
Index 67, NXTA adaptor  8. 3.- FACS bf 1K cells Zymo DNA purified
Index 68, NXTA adaptor  9. 4.- FACS bf 1K cells 800 ng RNA 
Index 69, NXTA adaptor  10. 4.- FACS bf 1K cells 400 ng RNA 
Index 70, NXTA adaptor  11. 4.- FACS bf 1K cells 100 ng RNA
Index 71, NXTA adaptor  12. 4.- FACS bf 1K cells 50  ng RNA
Index 72, NXTA adaptor  13. 1000 cells lysed w/out tspn

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 iaf2, iaf3, and NXTA indexes PCR_R.NXTAInd[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)



Results

  • Had leftover RNA generated from IVT Round 10, FACS sheath buffer testing with GM12878 which was done on live cells and testing FACS buffer as the media for cells instead of PBS. Samples were purified with AMPURE RNA beads, adding 30 uL to solution and 1:1.8X ratio of sample:beads.


  • RNA and Nuclease free water amounts used:
8/19/2013 800 ng H20 800 200 ng H2O 200 100 ng H20 100 50 ng H20 50
Index 61, N2 adaptor 1. PBS 1K cells 0.7460 9.2540 0.3730 9.6270 0.1865 9.8135
Index 62, N2 adaptor 2. PBS 12 uL 1000 cells 1.0600 8.9400 0.5300 9.4700 0.2650 9.7350
Index 63, N2 adaptor 3. FACS buffer 1K cells 0.7152 9.2848 0.3576 9.6424 0.1788 9.8212
Index 64, N2 adaptor 4. FACS buffer 1K cells (FORGOT TO ADD: 5 mM MgCl2) 2.117 7.883 0.5293 9.4707 0.2647 9.7353 0.1323 9.8677
Index 65, N2 adaptor 5. FACS buffer 12 uL 1000 cells 1.5562 8.4438 0.7781 9.2219 0.3890 9.6110
Index 66, N2 adaptor 6. 6 ng pure Jurkat DNA 0.4148 9.5852 0.2074 9.7926 0.1037 9.8963


  • quantification of RNA from TBU gel run during IVT round 10

File:ZhangLab 2 2013-08-15 13hr 39min-labeled.jpg





  • TBE gel after second strand synthesis and AMPURE DNA beads purification OR Zymo purification on samples 7 and 8
  • look as expected, similar to TBU gel on IVT RNA.
  • eluted samples in 20 uL N-H2O, concentrated to 10 uL, ran gel on 1 uL of the sample then.
  • qunaitation of samples:
' ng/ul intensity in 10 uL
Index 60, NXTA adaptor 1. PBS 1K cells 37.74099991 35733.80106 377.4099991
Index 61, NXTA adaptor 2. PBS 12 uL 1000 cells 23.86079158 22701.95068 238.6079158
Index 62, NXTA adaptor 3. FACS buffer 1K cells 19.98302232 19061.19057 199.8302232
Index 63, NXTA adaptor 4. FACS buffer 1K cells (FORGOT TO ADD: 5 mM MgCl2) 20.13495867 19203.84057 201.3495867
Index 64, NXTA adaptor 5. FACS buffer 12 uL 1000 cells 20.25003195 19311.88058 202.5003195
Index 65, NXTA adaptor 6. 6 ng pure Jurkat DNA 28.56915687 27122.54081 285.6915687
Index 66, NXTA adaptor 7. 1.- PBS 1K cells, Zymo DNA purified 9.466928681 9187.840274 94.66928681
Index 67, NXTA adaptor 8. 3.- FACS bf 1K cells Zymo DNA purified 18.21742479 17997.94054 182.1742479
Index 68, NXTA adaptor 9. 4.- FACS bf 1K cells 800 ng RNA 53.59111911 52174.73155 535.9111911
Index 69, NXTA adaptor 10. 4.- FACS bf 1K cells 400 ng RNA 30.9623625 30311.6409 309.623625
Index 70, NXTA adaptor 11. 4.- FACS bf 1K cells 100 ng RNA 6.663268348 6834.730204 66.63268348
Index 71, NXTA adaptor 12. 4.- FACS bf 1K cells 50 ng RNA 2.283038904 2602.710078 22.83038904
Index 72, NXTA adaptor 13. 1000 cells lysed w/out tsp


File:ZhangLab 2 2013-08-19 17hr 42min-labeled.jpg File:ZhangLab 2 2013-08-19 17hr 45min-labeled.jpg



  • qPCR from barcode addition. curves didn't go up since found out actually need to use 1X SYBE green, or .4X at the lowest. Not .075X which was used during this run.
  • samples in order
Samples:
Index 60, NXTA adaptor  1. PBS 1K cells
Index 61, NXTA adaptor  2. PBS 12 uL 1000 cells
Index 62, NXTA adaptor  3. FACS buffer 1K cells
Index 63, NXTA adaptor  4. FACS buffer 1K cells (FORGOT TO ADD: 5 mM MgCl2)
Index 64, NXTA adaptor  5. FACS buffer 12 uL 1000 cells
Index 65, NXTA adaptor  6. 6 ng pure Jurkat DNA
Index 66, NXTA adaptor  7. 1.- PBS 1K cells, Zymo DNA purified
Index 67, NXTA adaptor  8. 3.- FACS bf 1K cells Zymo DNA purified
Index 68, NXTA adaptor  9. 4.- FACS bf 1K cells 800 ng RNA 
Index 69, NXTA adaptor  10. 4.- FACS bf 1K cells 400 ng RNA 
Index 70, NXTA adaptor  11. 4.- FACS bf 1K cells 100 ng RNA
Index 71, NXTA adaptor  12. 4.- FACS bf 1K cells 50  ng RNA
Index 72, NXTA adaptor  13. 1000 cells lysed w/out tspn


File:2013-08-19 No Rnase III no PAP on cells, facs buffer testing.bmp



  • after custom nextera tagmentation and barcode addition

File:ZhangLab 2 2013-08-20 10hr 42min-labeled.jpg File:ZhangLab 2 2013-08-20 10hr 44min-labeled.jpg


  • amounts used for gel size selection
for 55 ng ' '
9.75270765 Index 60, NXTA adaptor 1. PBS 1K cells
10.09400317 Index 61, NXTA adaptor 2. PBS 12 uL 1000 cells
7.287737818 Index 62, NXTA adaptor 3. FACS buffer 1K cells
6.762720281 Index 63, NXTA adaptor 4. FACS buffer 1K cells (FORGOT TO ADD: 5 mM MgCl2)
11.85933263 Index 64, NXTA adaptor 5. FACS buffer 12 uL 1000 cells
5.952982882 Index 65, NXTA adaptor 6. 6 ng pure Jurkat DNA
4.889432272 Index 68, NXTA adaptor 9. 4.- FACS bf 1K cells 800 ng RNA
6.531546797 Index 69, NXTA adaptor 10. 4.- FACS bf 1K cells 400 ng RNA
11.87120643 Index 70, NXTA adaptor 11. 4.- FACS bf 1K cells 100 ng RNA
15.54802437 Index 71, NXTA adaptor 12. 4.- FACS bf 1K cells 50 ng RNA
55
ng total 550
sum 90.54969429
dye to add 18.10993886
per well 27.16490829


  • gel size cut validation

File:ZhangLab 2 2013-08-22 14hr 31min.jpg


  • gel size selection validation of size range
  • sample is R10, No RNIII No PAP, cells and facs buffer

File:ZhangLab 2 2013-08-22 18hr 06min-labeled.jpg