Brandon:LabNotes/Project1/2015-5-13
Naive and activated T cells from john chang Lab, pilot with Tn5-059 100 cell samples, mouse[edit]
- Guanidine HCl after transposition, taq2X and IVT on beads, 100 cell samples
- Previously tested Guanidine HCl after transposition, taq2X and IVT on beads, 100 cell samples with super mutant and it worked really well. with data as good as ENCODE DNase I data, but on 100 cells.
- doing pilot to make sure everything is working correctly with these cells. live naive T cells sorted from FACS.
- 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[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 N = naive A = activated Transposase is undiluted. 1X concentrated, using buffer D6. 1. t7-top2, 5X tn5-059, 100 cells, N1 rep1 2. t7-top2, 5X tn5-059, 100 cells, N1 rep2 3. t7-top2, 5X tn5-059, 100 cells, N2 rep1 4. t7-top2, 5X tn5-059, 100 cells, N2 rep2 5. t7-top2, 5X tn5-059, 100 cells, A1 rep1 6. t7-top2, 5X tn5-059, 100 cells, A1 rep2 7. t7-top2, 5X tn5-059, 100 cells, A2 rep1 8. t7-top2, 5X tn5-059, 100 cells, A2 rep2 9. t7-top2, 5X tn5-059, 1000 cells, N1 10. t7-top2, 5X tn5-059, 1000 cells, N2 11. t7-top2, 5X tn5-059, 1000 cells, A1 12. t7-top2, 5X tn5-059, 1000 cells, A2 13. t7-top2, 5X tn5-059, 6 ng pure DNA 14 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
cells in PBS and minutes amounts of sheath buffer from sorting
3 uL cells left, added 1 uL 4X LB
1.0 uL 5X Custom Tagmentation buffer 4.0 uL lysed cells/pure genomic DNA 1.0 uL T7 transposomes (MAKE SURE TO ADD LAST) (5X commercial concentration, tn5-059) ___________ 6.0 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 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.
Results[edit]
- TBU gel after IVT, Guanidine HCl to remove proteins
File:ZhangLab 2 2015-05-19 20hr 58min-labeled.jpg File:ZhangLab 2 2015-05-19 20hr 58min-invert-labeled.jpg
- second set of samples
File:ZhangLab 2 2015-05-19 21hr 02min-labeled.jpg File:ZhangLab 2 2015-05-19 21hr 03min-invert-labeled.jpg
conclusions[edit]
- 1000 cell samples are good. will repeat with 100 cell samples to try and get better amplification. will sort into PCR plate and carry out reactions in PCR plate so don't have to transfer cells to new tubes.