Brandon:LabNotes/Project1/2015-7-14

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RNA processing troubleshooting R5, new samples, new phos-sss_NPA_prmr[edit]


  • will also use a 5' phosphated sss_NPA_prmr this time, which is what was used before, but not in latest experiments. really doubt this is the problem.


  • maybe samples are bad, so using fresh GM12878 cells and IVT reactions, and will process the RNA as soon as it is ready.



Before starting protocols[edit]

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
1. idx49, t7-top2, 5X tn5-059, 500 cells rep1
2. idx50, t7-top2, 5X tn5-059, 500 cells rep2
3. idx51, t7-top2, 5X tn5-059, 6 ng pure DNA
4. idx52, t7-top2, 5X tn5-059, NTC



IVT Protocol[edit]

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

Generation of sc1-T7iBR-IdxXX transposomes

1. annealing of ME sequence to T7 transposon sequence

    • a. Make 100 uM stock solution of sc1-T7iBR-IdxXX and sc1-T7tspn-NoP-bot.
    • b. Incubate 2.5 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 1 minutes. Oligo's now at 10 uM in 25 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. Add 0.5 uL of 100% sterile glycerol to tube
2. Add 0.5 uL of annealed transposon to the 100% sterile glycerol and mix well
4. Add 1.0 uL of DILUTED Ez-TN5 transposase to well.
  • 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

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

1.0 uL 5X Custom Tagmentation buffer
2.0 uL lysed cells/pure genomic DNA
1.0 uL T7 transposomes (5X tn5-059, T7-top2)
1.0 uL N-H2O
___________
5.0 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, 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


Samples this time:

  • sample list
1. idx60, t7-top2, 5X tn5-059, 500 cells rep1
2. idx61, t7-top2, 5X tn5-059, 500 cells rep2
3. idx62, t7-top2, 5X tn5-059, 6 ng pure DNA
4. idx63, t7-top2, 5X tn5-059, NTC
5. idx64, 6/1/2015 6. t7-top2, 5X tn5-059, 1000 cells, N2
6. idx65, 6/1/2015 7. t7-top2, 5X tn5-059, 6 ng pure DNA


  • Amounts of RNA and water to add for MMLV RT (50 ng RNA)
' 3654.840109 5 ' ' ' '
2691.16008 2.5 in 10 uL uL RNA uL H20
1. idx49, t7-top2, 5X tn5-059, 500 cells rep1 30418.08091 74.42978997 744.2978997 0.671774031 10.82822597
2. idx50, t7-top2, 5X tn5-059, 500 cells rep2 46801.70139 116.9325398 1169.325398 0.427596972 11.07240303
3. idx51, t7-top2, 5X tn5-059, 6 ng pure DNA 205461.9161 528.5323707 5285.323707 /10 0.94601585 10.55398415
4. idx52, t7-top2, 5X tn5-059, NTC 1081.760032 -1.675141126 -16.75141126 1 10.5
6. t7-top2, 5X tn5-059, 1000 cells, N2 39365.57117 102.6939806 1026.939806 0.486883454 11.01311655
7. t7-top2, 5X tn5-059, 6 ng pure DNA 223827.8967 602.6731254 6026.731254 /10 0.829637127 10.67036287


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 11.5 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.
   4  uL 5X first strand buffer
   2  uL dNTP mix
   2  uL 100 mM DTT
  .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 5phos-sss_NPA_prmr primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since 5phos-sss_NPA_prmr has a high Tm.

a. Add 2.5 uL of 5phos-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, nxta_ME_B

1 uL custom tagmentation 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:
1. idx49, t7-top2, 5X tn5-059, 500 cells rep1
2. idx50, t7-top2, 5X tn5-059, 500 cells rep2
3. idx51, t7-top2, 5X tn5-059, 6 ng pure DNA
4. idx52, t7-top2, 5X tn5-059, NTC
5. idx53, 6/1/2015 6. t7-top2, 5X tn5-059, 1000 cells, N2
6. idx54, 6/1/2015 7. t7-top2, 5X tn5-059, 6 ng pure DNA
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[edit]

  • total RNA.
7/15/15 RNA amounts to use 3654.840109 5 ' ' ' '
2691.16008 2.5 in 10 uL uL RNA uL H20
1. idx49, t7-top2, 5X tn5-059, 500 cells rep1 30418.08091 74.42978997 744.2978997 0.671774031 10.82822597
2. idx50, t7-top2, 5X tn5-059, 500 cells rep2 46801.70139 116.9325398 1169.325398 0.427596972 11.07240303
3. idx51, t7-top2, 5X tn5-059, 6 ng pure DNA 205461.9161 528.5323707 5285.323707 /10 0.94601585 10.55398415
4. idx52, t7-top2, 5X tn5-059, NTC 1081.760032 -1.675141126 -16.75141126 1 10.5
6. t7-top2, 5X tn5-059, 1000 cells, N2 39365.57117 102.6939806 1026.939806 0.486883454 11.01311655
7. t7-top2, 5X tn5-059, 6 ng pure DNA 223827.8967 602.6731254 6026.731254 /10 0.829637127 10.67036287
  • TBU gel after IVT, Guanidine HCl to remove proteins

File:ZhangLab 2 2015-07-15 10hr 41min-labeled.jpg

  • qPCR curves

File:2015-07-15 nxta me b trbleshoot, new smpl R5.bmp


  • after adding barcodes, TBE gel

File:ZhangLab 2 2015-07-15 19hr 07min-labeled.jpg


  • gel size selection amounts
7/15 gel size selection 7520.400224 5 ' '
4596.570137 2.5 for 60 ng
1. idx60, t7-top2, 5X tn5-059, 500 cells rep1 31126.30093 25.18405653 2.382459709 4.764919419
2. idx61, t7-top2, 5X tn5-059, 500 cells rep2 40901.84122 33.54256232 1.78877211 3.577544221
3. idx62, t7-top2, 5X tn5-059, 6 ng pure DNA 47326.41141 39.03584511 1.537048829 3.074097657
4. idx63, t7-top2, 5X tn5-059, NTC 2471.710074 0.683153603 1 2
5. idx64, 6/1/2015 6. t7-top2, 5X tn5-059, 1000 cells, N2 32982.67098 26.77133246 2.24120335 4.4824067
6. idx65, 6/1/2015 7. t7-top2, 5X tn5-059, 6 ng pure DNA 43672.2613 35.9113902 1.670779095 3.341558189
  • gel size selection, 220-800 ish bp

File:ZhangLab 2 2015-07-15 20hr 15min-labeled.jpg


  • gel size selection validation

File:ZhangLab 2 2015-07-16 12hr 31min-labeled.jpg


conclusions[edit]

  • submitted for sequencing