Brandon:LabNotes/Project1/2013-11-21

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Round 14, single cell analysis Nextera and THS[edit]

  • trying out IVT assay and nextera assay on single cells. GM12878 Cells are sorted with FACS into lo-bind 200 ul PCR tubes at the stem cell core, with gating to isolate live cells.


  • samples
    • Single cell samples (5 samples, with transposome dilutions)
    • Samples done with Nextera
    • 2 1000 cell samples
    • pure DNA
    • NTC


  • samples
1. nextera single cell rep1 (1:10 dilution)
2. nextera single cell rep2 (1:10 dilution)
3. nextera single cell rep3 (1:10 dilution)
4. nextera single cell rep4 (1:10 dilution)
5. nextera single cell rep5 (1:50 dilution)
6. nextera single cell rep6 (1:50 dilution)
7. nextera single cell rep7 (1:50 dilution)
8. nextera 50 cells (1:10 dilution)
9. nextera 100 cell (1:10 dilution)
10. nextera 1000 cell (1:10 dilution)
11. nextera pure Jurkat DNA (1:10 dilution)
12. nextera NTC
13. IVT single cell rep1
14. IVT single cell rep2, 4X diluted tspsome
15. IVT single cell rep3, 4X diluted tspsome
16. IVT single cell rep4, 8X diluted tspsome
17. IVT single cell rep5, 8X diluted tspsome
18. IVT single cell rep6, 16X diluted tspsome
19. IVT single cell rep7, 16X diluted tspsome
20. IVT 50 cell, 4X diluted tspsome
21. IVT 100 cell, 4X diluted tspsome
22. IVT 1000 cell 
23. IVT pure Jurkat DNA
24. IVT NTC



Nextera Tagmentation Protocol[edit]

1. See step 3 in IVT protocol. Do the cell lysis section for the "nextera tagmentation samples", samples 1-12


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


adapted from andrews PCR procotol for nextera, which is the basic nextera procotol. barcodes added first, followed by amplification of those barcodes.


4. First step of 2-step PCR thermocycling, run for 7 cycles. barcode addition

barcodes to use. samples
Indx 49, NXTA adaptor, 1. nextera single cell rep1
Indx 50, NXTA adaptor, 2. nextera single cell rep2
Indx 51, NXTA adaptor, 3. nextera single cell rep3
Indx 52, NXTA adaptor, 4. nextera single cell rep4
Indx 53, NXTA adaptor, 5. nextera single cell rep5
Indx 54, NXTA adaptor, 6. nextera single cell rep6
Indx 55, NXTA adaptor, 7. nextera single cell rep7
Indx 56, NXTA adaptor, 8. nextera 50 cells
Indx 57, NXTA adaptor, 9. nextera 100 cell
Indx 58, NXTA adaptor, 10. nextera 1000 cell
Indx 59, NXTA adaptor, 11. nextera pure Jurkat DNA
Indx 60, NXTA adaptor, 12. nextera NTC
6   ul Tagmentation reaction
7   ul KAPA SYBR FAST qPCR mix
.6   ul Adaptor 1 (10uM)
.6   ul XX Index primer per sample (10uM)
1 ul H2O 
----------------
15.2 uL total


PCR cycling. 72C for 3 minutes is for gap fill in.

72C 3min -> 95C 10 sec -> (95C 15sec -> 62C 30 sec -> 72 45sec) x 7 -> 4C forever
Monitor the reactions on a real-time thermal cycler and terminate them before the curves reach saturation.


5. Second Round of PCR, amplification


add the following to above dry tube:
15 uL first reaction
15 uL KAPA SYBR FAST
2  uL primer 1
2  uL of primer 2
15 uL nuclease free H2O
---------------
45 total per reaction

PCR cycling for addition of index primer
10 cycles max
95C 1 min -> 95C 30 sec -> (95C 10sec -> 60C 30 sec -> 72C 1 min) x 5 -> 72C 3min.

  • 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. 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:
  • sample list
13. IVT single cell rep1
14. IVT single cell rep2, 4X diluted tspsome
15. IVT single cell rep3, 4X diluted tspsome
16. IVT single cell rep4, 8X diluted tspsome
17. IVT single cell rep5, 8X diluted tspsome
18. IVT single cell rep6, 16X diluted tspsome
19. IVT single cell rep7, 16X diluted tspsome
20. IVT 50 cell, 4X diluted tspsome
21. IVT 100 cell, 4X diluted tspsome
22. IVT 1000 cell 
23. IVT pure Jurkat DNA
24. IVT NTC


CELL WASHING - (wash cells with PBS)

  • 1. count cells, spin down all cells at 250-500 g for 5 minutes
  • 2. wash with 1X PBS, centrifuge 250-500 g for 5 minutes, resuspend to XXXX cells/uL
  • 3. Aliquot cells to sample tubes

CELL LYSIS

  • 1. dilute LB to 2X, or as needed (from 10X stock)
  • 2. refrigerate or warm LB or keep at RT
  • 3. add lysis buffer to cell aliquots, mix briefly (mineral oil optional)
  • 4. transposition ready


LYSIS BUFFER NOTES

  • Lysis buffer is: 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.


4. transposition reaction, using (T7tspn-top2)


SAMPLES (ALL), 55c 10 MINUTES

  • 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)
1.4 uL Nuclease free water
.6 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL)
OR DILUTED TSPN, 4X, 8X, 16X
___________
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)


CLEAN WITH ZYMO RNA CLEAN AND CONCENTRATOR NOW

8. Clean RNA with AMPURE RNA beads. elute in 20 uL.

  • 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.
    • elute in 20 uL and concentrate to 10 uL
  • can quantitate with Qubit or on TBU gel.


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


Samples this time:

Samples:
  • sample list
1. 500 nuclei 37C 30 rep1
2. 500 nuclei 55C rep1
3. 500 cells clysis 37C 30 rep1
4. 500 cells clysis 55C rep1
5. pure jurkat DNA 3 ng
6. NTC


  • Amounts of RNA and water to add for MMLV RT (50 ng RNA)


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 20 uL and concentreate . (can also use qiaquick)

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 1X FINAL CONCENTRATION IN SOLUTION

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

Samples:
1. 500 nuclei 37C 30 rep1
2. 500 nuclei 55C rep1
3. 500 cells clysis 37C 30 rep1
4. 500 cells clysis 55C rep1
5. pure jurkat DNA 3 ng
6. NTC
KAPA SYBR FAST qPCR mix until saturation, X35 cycles

9  uL Taq2X
2  uL primers, 2 uL F, (T7-top2-PCR-iaf, OR iaf2, iaf3)
2  uL NXTA indexes PCR_R.NXTAInd[XX], SPECIFIC TO EACH SAMPLE
4  uL H2O
1  uL 25X SYBR green
12 uL DNA template (use half RT reaction)
_____________
30 uL

KAPA SYBR cycles:
95C 3min, (95C for 30s, 60C for 30s, 72C for 2 min) X35, 72C for 5 min, 4C forever

  • terminate before curves saturate (usually cycle 6-8)



16. Gel Size selection

  • gel size select from 400-800 bp, follow gel size selection protocol
  • do not need to include controls.



results[edit]

NEXTERA TAGMENTATION RESULTS

  • After PCR amplification. 5 uL of sample used for the gel.

File:ZhangLab 2 2013-11-22 14hr 24min-labeled.jpg File:ZhangLab 2 2013-11-22 14hr 27min-labeled.jpg


  • DNA quantification results
  • ran 5 uL on gel. Thus total amount/5 is true amount
' calc ng/ul true ng/ul intensity '
11/22/2013 5 2673.070427
2.5 /5 1641.175489 30 ng
1. nextera single cell rep1 12.64317696 2.528635392 5827.852674 2.372821332
2. nextera single cell rep2 55.45371951 11.0907439 23498.24553 2.704958321
3. nextera single cell rep3 2.355602366 0.471120473 1581.574214 63.67797986
4. nextera single cell rep4 14.05410835 2.810821671 6410.225858 10.67303569
5. nextera single cell rep5 5.029724004 1.005944801 2685.339247 29.82270993
6. nextera single cell rep6 10.44931135 2.08986227 4922.317147 14.35501297
7. nextera single cell rep7 8.587947706 1.717589541 4154.024457 17.46633831
8. nextera 50 cells 19.78398744 3.956797489 10316.47569 7.581889163
9. nextera 100 cell 32.12347103 6.424694206 16119.20894 4.669482942
10. nextera 1000 cell 55.49915515 11.09983103 27111.79696 2.702743845
11. nextera pure Jurkat DNA 53.28474744 10.65694949 26070.45539 2.815064483
12. nextera NTC 8.943341043 1.788668209 5218.581694 16.77225539


  • nextera gel size selection

File:ZhangLab 2 2013-11-22 17hr 33min-labeled.jpg


  • nextera gel size selection validation
  • accidentally used 5X less DNA then needed and smear is too weak. thus will repeat gel size selection

File:ZhangLab 2 2013-11-25 10hr 59min-labeled.jpg


  • nextera gel size selection repeat

File:ZhangLab 2 2013-11-26 10hr 54min-labeled.jpg


  • nextera gel size selection repeat

File:ZhangLab 2 2013-11-26 15hr 27min-labeled.jpg





THS tagmentation/IVT amplification results


  • RNA quantification results
  • 50 cell sample generated enough RNA for downsteam processing. could use this as a minimum.
' ng/ul intensity '
5 1843.006801
2.5 1153.916376 in 10 ul
8. IVT 50 cells 8.064298707 2687.63836 80.64298707
9. IVT 100 cell 22.98520591 6800.380075 229.8520591
10. IVT 1000 cell 100.0102727 28031.27448 1000.102727
11. IVT pure Jurkat DNA 130.8919219 36543.374 1308.919219
12. IVT NTC -2.538743809 -234.9436694 -25.38743809
-1.633384745 14.60603547 -16.33384745


  • RNA after ~16.5 hours IVT, run on TBU gel
  • single cell amplifications for IVT appear to not have worked. This is expected.

File:ZhangLab 2 2013-11-22 12hr 12min-labeled.jpg File:ZhangLab 2 2013-11-22 12hr 16min-labeled.jpg


  • qPCR curves for adding barcodes. samples are in order presented.
  • used T7-top2-PCR-iaf2, and thus curves are working and better.
  • Stopped at end of 6th cycle, since that is what was done for other assays. in future can see when curves saturate.
Samples:


  • first PCR cycling. ran for 6 cycles


  • amounts used for gel size selection


  • TBE gel size selection validation


future and conclusions[edit]

  • Nextera single cell is benchmark to compare to THS
    • low amounts of total unique reads, and high clonal rate and this is with very few total reads
    • also has a high fail map rate
    • gel results have same intensity as NTC. Thus cannot be sure of background amplification


  • Next time will try and sort single cells into lysis buffer that has a bubble in it. Otherwise minimum volume should be 3 uL to capture the cells.


  • 50 cell sample generated enough RNA for downstream processing in the IVT samples. could use this as a minimum for multiplexing single cells


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