Brandon:LabNotes/Project1/2015-6-9

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Experimental Samples, day 7 Tsle (short lived effector), day 7 Tmp (memory precursor) john chang Lab, Tn5-059 100 cell samples, mouse

  • T-cell samples
    • day 7 Tsle (short lived effector)
    • day 7 Tmp (memory precursor)


  • Guanidine HCl after transposition, taq2X and IVT on beads, 100 cell samples



  • Need to collect cells in 96 well plate or 0.2 mL tubes in higher volumes. Then spin down plate at 200Xg for 4 minutes, pipet out excess PBS/sheath buffer and leave 2-3 uL at the bottom without touching with pipet. Add 4X LB to 3 uL sample.


Before starting protocols

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • sc1 transposomes
  • custom 3' transpson
  • IVT reagents
  • cells etc
  • 5X, 2X taq polymerase
  • Zymo DNA clean and concentrator


  • 2. Samples
samples
Transposase is undiluted. 1X concentrated, using buffer D6.
1. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle
2. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle
3. t7-top2, 5X tn5-059, 1000 cells, day 7 Tsle
4. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle
5. t7-top2, 5X tn5-059, 100 cells, day 7 Tme
6. t7-top2, 5X tn5-059, 100 cells, day 7 Tme
7. t7-top2, 5X tn5-059, 250 cells, day 7 Tme
8. t7-top2, 5X tn5-059, 100 cells, day 7 Tme
9. t7-top2, 5X tn5-059, 6 ng pure DNA
10. t7-top2, 5X tn5-059, NTC


IVT Protocol

  • Using Tn5-059 t7-top2 generated by Trina, at 5X commercial concentration. Dilute 25X transposome solution with tn5 storage solution provided by Trina.

3. Prepare samples, lyse cells with lysis buffer

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 XX cells/uL
  • 3. Count cells again, aliquot cells to sample tubes

CELL LYSIS

  • 1. dilute Lysis Buffer (LB) to 2X, or as needed (from 10X stock)
  • 3. add LB 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 Add all components and incubate at 37C FOR 30 MINUTES

cells in PBS and minutes amounts of sheath buffer from sorting

3 uL cells left, added 1 uL 4X LB

1.2 uL 5X Custom Tagmentation buffer
4.0 uL lysed cells/pure genomic DNA
1.2 uL T7 transposomes (MAKE SURE TO ADD LAST) (5X commercial concentration, tn5-059)
___________
6.4 uL total solution


5. Bring to 15 uL with N-H2O. Add 15 uL 8M guanidine HCl for a total of 30 uL.

    • If using beads next add 1.8X (54 uL) beads per sample


ELUTE IN 9.6 or 12 uL DEPENDING, leave beads in solution


6. Fill in reaction

  • Add 2.4 uL of 5X taq polymerase or 3.0 uL of taq2X. Run at 72C for 3 minutes. (same as nextera)


8. 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 10 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)



9. Clean with Zymo clean and concentrator

  • elute samples in 10 uL of N-H2O
  • quanitate with Qubit or on TBU gel.



AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO


UPDATE 3/25/2015 CONTINUING TO RNA PROCESSSING TO GENERATE SEQUENCING LIBRARIES


Samples this time:

  • sample list


  • 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.


d. Add polymerases etc to reactions

          taq2X: Add  5.875 uL, Incubate at 72C for 8 minutes
          
            NTC: Just add 1 uL of Klenow exo-


11. Clean DNA with AMPURE beads depending. elute in 20 uL and concentreate. (can also use qiaquick)

  • USED AMPURE DNA BEADS AT 1:1.8X RATIO
  • add 45 uL N-H2O
  • add 135 uL beads per reaction
  • elute on 20 uL

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.

RAN FOR 16 MINUTES


12. Fragmenting and 3' End tagging with Custom Nextera transposome USED EZ-TN5 AND BUFFER D6

1 uL nextera LMW buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
X uL Nuclase free H2O
1 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 6 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)


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

Samples:
  • sample list


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) USED iaf2
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:
72C 3 m, 95C 30s, (95C for 10s, 63C for 30s, 72C for 3 min) X15, 72C for 3 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

  • total RNA.


  • TBU gel after IVT, Guanidine HCl to remove proteins


conclusions

  • blah