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==Guanidine HCl on cells, using R2 f2 isolated transposase== *R2 f2 had the best results in terms of [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2014-9-15 contamination with nucleases and ability to perform IVT]. Thus till try it out on cells and combining samples. *8x diluted tranposase seems stringent enough to dilute out any nucleases and give a good IVT smear. samples -all using T7-top2 tspn, 1-10 for pooling. using buffer D6. 1. 100 cells GM12878, 8X dil R2 f2 rep1 2. 100 cells GM12878, 8X dil R2 f2 rep2 3. 100 cells GM12878, 8X dil R2 f2 rep3 4. 100 cells GM12878, 8X dil R2 f2 rep4 5. 100 cells GM12878, 8X dil R2 f2 rep5 6. 100 cells GM12878, 8X dil R2 f2 rep6 7. 100 cells GM12878, 8X dil R2 f2 rep7 8. 100 cells GM12878, 8X dil R2 f2 rep8 9. 100 cells GM12878, 8X dil R2 f2 rep9 10. 100 cells GM12878, 8X dil R2 f2 rep10 11. 1000 cells GM12878, 8X dil R2 f2 12. 500 cells GM12878, 8X dil R2 f2 13. 6 ng Jurkat DNA, 8X dil R2 f2 ===IVT Protocol=== *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) 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 2X Lysis buffer 1.0 uL genomic DNA/cells 1.0 uL 5X Custom Tagmentation buffer 1.0 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) 1.0 uL N-H20 ___________ 5.0 uL total solution 5. Add Guanidine HCl. Add 5 uL guanidine HCl. pool samples if needed. samples that don't need to be pooled, bring to 15 uL with N-H20, then add 15 uL 8M guanidine HCl **If using beads next add 1.8X (54 uL) beads or for sample, or for pooled samples 96 uL beads. '''ELUTE IN 9.6 or 12 uL DEPENDING, LEAVE BEADS IN REACTION''' 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''' '''Results''' *TBU after IVT *pooling didnt work, maybe because nucleases got to them before hand since it was a small amount of DNA. Or maybe this method won't work to well with small cell numbers. [[File:ZhangLab 2 2014-09-18 17hr 00min-labeled.jpg|600px]]
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