Brandon:LabNotes/Project1/2014-5-23
Pooling and purification with AMPURE beads on 100 cell samples (IVT not run on beads)[edit]
- In previous experiments controls did not work with cell samples, maybe since beads were left in solution. will add elution buffer to beads, and heat at 55C for 2 minutes, then elute from beads and see if cell samples amplify when IVT is NOT performed on beads.
- where control cell samples did not work
- In 4/30 Testing pooling and purification with AMPURE beads
- In 3/11 Testing pooling and purification with AMPURE beads
- In 5/21 Pooling and purification with AMPURE beads on FACS sorted 10 cell samples
- using 5X diluted transposase to save transposase. and appeared to work well with transposase dilution testing with 100 cells
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
- pool samples 1-30.
1. sc1-T7iBR-Idx25 puf., 100 cells 2. sc1-T7iBR-Idx26 puf., 100 cells 3. sc1-T7iBR-Idx27 puf., 100 cells 4. sc1-T7iBR-Idx28 puf., 100 cells 5. sc1-T7iBR-Idx29 puf., 100 cells 6. sc1-T7iBR-Idx30 puf., 500 cells 7. sc1-T7iBR-Idx31 puf., 1000 cells 8. sc1-T7iBR-Idx26 puf., 3 ng Jurkat DNA 9. sc1-T7iBR-Idx25 puf., NTC
IVT Protocol[edit]
- If need to make more transposome, do first 2 steps. If not goto step 3.
MAKE 2X DILUTED TRANSPOSASE 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) illuminia adaptor transposome
1. annealing of ME sequence to Nextera transposon sequence
- a. Make 100 uM stock solution of (sc1-T7tspn-NoP-bot, "pMENTS") and (sc-ILA-adaptor).
- b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 1 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 (or .6 uL undiluted) prepared T7 transposomes (MAKE SURE TO ADD LAST) (5X diluted) ___________ 4.0 uL total solution
5. Protease digestion of transposase, protease inactivation
To each tube, add: 1.0 uL Qiagen Protease, use 1 uL of 11.1 ug/uL (.5 AU/ml) protease (dilute 5.5 uL 20 ug/ul with 4.5 uL N-H2O) Incubate: 50C 10 minutes, 70C 20 minutes
6. pooling and ampure beads purification
a. Use ampure beads at 1.8X (8 uL pre rxn) b. follow AMPURE beads cleaning protocol (one wash, 2 EtOH washes) c. elute at 55C on 9.6 uL H2O (remove elution buffer from beads)
7. 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)
ADDED 2.2 uL taq 5X polymerase
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
Results[edit]
- TBU gel after IVT
- pooled, 500 and 1000 cell samples did not really work.
File:ZhangLab 2 2014-05-24 12hr 36min-labeled.jpg
conclusions[edit]
- pooled, 500 and 1000 cell samples did not really work. not sure why, but this is the fourth time this has happened. maybe it is because ampure beads to not really denature proteins, since it does not have guanidine HCl which is stated in this blog about ampure beads.
- Maybe protease digestion is not sufficient and need a denaturing agent such as Guanidine HCl.
- will try using columns and EtOH preciptation. zymo wash buffer in DNA clean and concentrator has guanidine HCl in it to denature proteins.