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==Testing pooling and purification with AMPURE beads== *In [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2014-3-11 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 try BOTH tagmentation, protease digestion, taq2X fill in then AMPURE beads purification, AND tagmentation, protease digestion, AMPURE beads purification, then taq2X fill in, to see if there are any IVT amplification efficiency differences. Optimal is taq2x fill in after AMPURE beads so tagemtantion reaction volumes can remain smaller. **doing protease digestion to ensure there is no barcode cross contamination by insertion into other samples upon pooling. *P1= tagmentation, protease digestion, taq2X fill, AMPURE beads purification *P2= tagmentation, protease digestion, AMPURE beads purification, then taq2X fill in *C1= combined group1 *C2= combined group2 *will dilute transposase 2X to save transposase. *Can also test: 1. combining individually barcoded samples and running a large IVT reaction 2. using guandine HCL for proteins denatureation then purification ===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 1. sc1-T7iBR-Idx25 puf., 80 cells, C1, P1 2. sc1-T7iBR-Idx26 puf., 80 cells, C1, P1 3. sc1-T7iBR-Idx27 puf., 80 cells, C1, P1 4. sc1-T7iBR-Idx28 puf., 80 cells, C1, P1 5. sc1-T7iBR-Idx29 puf., 80 cells, C1, P1 6. sc1-T7iBR-Idx25 puf., 3 ng pure DNA, P1 7. sc1-T7iBR-Idx25 puf., 80 cells, C2, P2 8. sc1-T7iBR-Idx26 puf., 80 cells, C2, P2 9. sc1-T7iBR-Idx27 puf., 80 cells, C2, P2 10. sc1-T7iBR-Idx28 puf., 80 cells, C2, P2 11. sc1-T7iBR-Idx29 puf., 80 cells, C2, P2 12. sc1-T7iBR-Idx30 puf., 1000 cells, C2, P2 13. sc1-T7iBR-Idx31 puf., 3 ng pure DNA, C2, P2 14. sc1-T7iBR-Idx32 puf., NTC, C2, P2 ===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 '''USED 80 CELLS INSTEAD''' Add all components and incubate at 37C FOR 30 MINUTES 0.5 uL 5X Custom Tagmentation buffer 1.5 uL lysed cells/pure genomic DNA 0.5 uL (or .6 uL undiluted) prepared T7 transposomes (MAKE SURE TO ADD LAST) ___________ 2.5 uL total solution 5. Protease digestion of transposase, protease inactivation To each tube, add: .5 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 '''SAMPLES 7-13 EACH GET SOME ~5.4 uL AMPURE BEADS, THEN POOL 7-11 AND GO STRAIGHT TO AMPURE BEADS PUF.''' '''SAMPLES 1-6 UNDERGO TAQ2X FILL IN ETC (NORMAL PROTOCOL)''' 6. Fill in reaction *Add .75 uL of 5X taq polymerase or 3.0 uL of taq2X. Run at 72C for 3 minutes. (same as nextera) '''samples 1-5 pooled, 6 kept separate''' '''SAMPLES 7-14 PERFORM TAQ2X FILL IN REACTION''' '''Using AMPURE RNA beads since they are verified as RNase etc free''' 7. Pool samples. Clean with RNA AMPURE beads a. pool samples. add 1.5X volumes of N-H2O to each sample With 12 uL sample volume: i. add 18 uL N-H2O ii. add 54 uL beads (1.8X) b. follow AMPURE beads cleaning protocol (one wash, 2 EtOH washes) c. resuspend in 12 uL H2O (keep elution buffer on beads) d. proceed to IVT 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=== *quantitation of gel smears {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''intensity''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||5||1910.480057|| |- | ||2.5||1175.520035||in 10 uL (ng) |- | 7-11. sc1-T7iBR-Idx25-29 puf., 80 cells, C2, P2||20.72276042||6532.720195||207.2276042 |- | 6. sc1-T7iBR-Idx25 puf., 3 ng pure DNA, P1||14.07709263||4579.000136||140.7709263 |- | 13. sc1-T7iBR-Idx31 puf., 3 ng pure DNA, C2, P2||261.0885967||77196.4323||2610.885967 |- | |} *TBU gel after IVT [[File:ZhangLab 2 2014-05-01 16hr 22min-labeled.jpg|600px]] '''UPDATE 5/15/2014''' *Ran sample 7-11. through RNA processing and gel result is there. [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2014-5-13 Optimizing PCR for 3' adaptor addition, RNA processing and pooled sample processing] *did gel size selection on it and results are there ===conclusions=== *looks like washing samples with AMPURE beads, then fill in with taq2X worked. *peforming AMPURE beads after taq2X fill in did not work '''PROBABLY BECAUSE 1.5X WATER WAS ADDED TO SAMPLES BEFORE AMPURE BEADS PURIFICATION IN MARCH, AND WAS NOT THIS TIME'''. however back in march [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2014-3-11 AMPURE beads pooling after taq2x] it appeared to work inefficiently. though samples were not purified after. It could be samples got lost during purificaiton, which can be checked since flow through was saved. *can try and increase yield, and use guanidine chloride in reaction. *can try guadine chlorid and skip protase digestion, followed by ampure beads purification. can see if yields are increased. *normally ~40-60 million bp are accessible. this is 60/3000 = 2% of the genome and thus is approximately .12 pg per cell, if the genome of a human cell is 6 pg total. **Thus in this experiment, 400 cells were used, 2.4 ng of total DNA, 48 pg of accessible DNA. 207 ng of product was obtained, thus amplification was 4312X. **50 cell is 6 pg of accessible DNA.
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