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==Pooling and purification with AMPURE beads on FACS sorted 10 cell samples== *previous experiments seem to have worked, and thus will try on 10 cells sorted with FACS. used GM12878, along with controls. will run 30 samples of 10 cells each thus pooled sample will be 300 cells worth of material. *In [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2014-4-30 4/30 Testing pooling and purification with AMPURE beads] smear was seen with 5 indexes of 80 cells each. *In [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2014-3-11 3/11 Testing pooling and purification with AMPURE beads] it was found to contain the most IVT product after amplification when compared to EtOH purification or column purification. Also it even though there was only very little amplification from IVT from pooling it still gave IVT product when the transposome reaction buffer was not optimized, while purification with other methods did not. *will dilute transposase 5X to save transposase. and appeared to work well with [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2014-2-26 transposase dilution testing with 100 cells] ===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 *pool samples 1-30. 1. sc1-T7iBR-Idx25 puf., 10 cells 2. sc1-T7iBR-Idx26 puf., 10 cells 3. sc1-T7iBR-Idx27 puf., 10 cells 4. sc1-T7iBR-Idx28 puf., 10 cells 5. sc1-T7iBR-Idx29 puf., 10 cells 6. sc1-T7iBR-Idx30 puf., 10 cells 7. sc1-T7iBR-Idx31 puf., 10 cells 8. sc1-T7iBR-Idx32 puf., 10 cells 9. sc1-T7iBR-Idx33 puf., 10 cells 10. sc1-T7iBR-Idx34 puf., 10 cells 11. sc1-T7iBR-Idx35 puf., 10 cells 12. sc1-T7iBR-Idx36 puf., 10 cells 13. sc1-T7iBR-Idx37 puf., 10 cells 14. sc1-T7iBR-Idx38 puf., 10 cells 15. sc1-T7iBR-Idx39 puf., 10 cells 16. sc1-T7iBR-Idx40 puf., 10 cells 17. sc1-T7iBR-Idx41 puf., 10 cells 18. sc1-T7iBR-Idx42 puf., 10 cells 19. sc1-T7iBR-Idx43 puf., 10 cells 20. sc1-T7iBR-Idx44 puf., 10 cells 21. sc1-T7iBR-Idx45 puf., 10 cells 22. sc1-T7iBR-Idx46 puf., 10 cells 23. sc1-T7iBR-Idx47 puf., 10 cells 24. sc1-T7iBR-Idx48 puf., 10 cells 25. sc1-T7iBR-Idx49 puf., 10 cells 26. sc1-T7iBR-Idx50 puf., 10 cells 27. sc1-T7iBR-Idx51 puf., 10 cells 28. sc1-T7iBR-Idx52 puf., 10 cells 29. sc1-T7iBR-Idx53 puf., 10 cells 30. sc1-T7iBR-Idx54 puf., 10 cells 31. sc1-T7iBR-Idx55 puf., 500 cells 32. sc1-T7iBR-Idx56 puf., FACS NTC (sheath buffer run thru facs) 33. sc1-T7iBR-Idx25 puf. diluted 2X ~ .5 uL tspsome added, 3 ng Jurkat DNA 34. sc1-T7iBR-Idx26 puf. diluted 2X ~ 1 uL tspsome added, 1000 cells ===IVT Protocol=== *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 0.9 uL 5X Custom Tagmentation buffer 1.5-2.0 uL lysed cells/pure genomic DNA 0.9 uL (or .6 uL undiluted) prepared T7 transposomes (MAKE SURE TO ADD LAST) (5X diluted) ___________ 3.3 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 b. follow AMPURE beads cleaning protocol (one wash, 2 EtOH washes) c. elute in 10 uL H2O (keep on beads for IVT) '''ELUTED INTO 8 uL BY ACCIDENT''' '''pool samples 1-30,32 (i25-i54,i56). clean each tube with 251.1 uL beads, and mix well when adding to pooled solution at end. added 50 uL water and 90 uL beads extra to pooled rxn before purifiying''' '''samples 31,33,34 kept separate and undergo ampure beads purifying. added 1.5X water before purifying''' 7. Fill in reaction *Add 2.0 uL of 5X taq polymerase or 3.0 uL of taq2X. Run at 72C for 3 minutes. (same as nextera) '''ADDED 2.7 TO POOLED SAMPLE SINCE BEADS ADDED ELUTION VOLUME.''' 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) '''ADDED 10 UL IVT MIX TO POOLED SAMPLE SINCE INCREASED VOLUME''' 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=== *TBU gel after IVT [[File:ZhangLab 2 2014-05-22 17hr 36min-labeled.jpg|600px]] ===conclusions=== *After looking at samples, using this method on 500 cells and 1000 cells has with IVT on beads has not worked in [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2014-4-30 4/30 Testing pooling and purification with AMPURE beads] and [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2014-3-11 3/11 Testing pooling and purification with AMPURE beads]
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