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==D6 versus TA buffer tagmentation buffer for accessibility, with NEB T7 Hiscribe for IVT== *last time tagmentation buffer was titrated it was with a different transposon and different IVT kit and slightly different protocol. will test again to see which is more efficient now. *now using scheme #6 for RNA processing after 2x stranded cDNA generated. [[File:2015-10-02 experiemnts summary.png|600px]] ===Before starting protocols=== 1. Check if have enough reagents etc for the protocol *lysis buffer *tn5059 transposomes *custom 3' transpson *IVT reagents *cells etc *5X, 2X taq polymerase *Zymo DNA clean and concentrator *2. Samples samples 1. i502_T7-1_short-i701_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep1 2. i502_T7-1_short-i702_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep2 3. i502_T7-1_short-i703_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep3 4. i502_T7-1_short-i704_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep1 5. i503_T7-1_short-i701_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep2 6. i503_T7-1_short-i702_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep3 7. i503_T7-1_short-i703_long, T7-nXTv2_i5-1, 5X tn5059, 6ng DNA, buffer D6 8. i503_T7-1_short-i704_long, T7-nXTv2_i5-1, 5X tn5059, NTC, buffer D6 ===IVT Protocol=== *If need to make more transposome, do first 2 steps. If not goto step 3. '''Generation of transposomes''' 1. annealing of ME sequence to T7 transposon sequence a. Make 100 uM stock solution of tranposon and ME only bottom end. b. Incubate 15 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 2 minutes, then cool to 14C at 0.1 C/s. c. Oligo's now at 30 uM in 50 uL. 2. Transposome complex generation, run controls!!! *add the below components into one tube and incubate for 30 minutes at RT a. Add 1.0 uL of 100% sterile glycerol to tube b. Add 1.0 uL of annealed transposon to the 100% sterile glycerol and mix well c. Add 2.0 uL of transposase to well. *store at -20, is good for a year 3. Prepare samples, lyse cells with lysis buffer CELL WASHING - (wash cells with PBS) a. count cells, spin down all cells at 250Xg for 5 minutes '''80 uL 50X LB, 920 uL protease inhbiitors''' b. wash with 1X PBS, centrifuge 250Xg for 5 minutes, resuspend to 33 cells/uL c. Count cells again to be sure of cells/uL number CELL LYSIS a. dilute Lysis Buffer (LB) to 4X add protease inhibitors, or as needed (from 10X stock) b. add 3 uL cells to each well in 96 well plate c. add 1 uL 4X LB + protease inhibitors to each well, mix briefly d. 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 '''Added in 3 uL cells, then 1 uL 4X LB''' 1.2 uL 5X Custom Tagmentation buffer 4.0 uL lysed cells/pure genomic DNA 1.2 uL T7 transposomes (5X tn5-059, indexed Tsome as listed) ___________ 6.4 uL total solution 5. Stop reaction by adding (4.0 uL) 50 mM EDTA. (20 mM final EDTA) Incubate for 15 mins at 37C. 6. Nuclei pooling and recovery '''did not do in this run''' a. Pool all nuclei into one tube b. Add 8 uL PBS to each well of the 96 well plate, and add to pooled tube c. Mix pooled tube gently and throughly then aliquot 25 nuclei, 5.25 uL to each well in 96 well plate (9600/2016) = 4.76 nuclei/uL; 25/4.76 = 5.25 *sort with FACS? can sort into 10 uL EB buffer. *In paper: nuclei were first stained with DAPI (Invitrogen) at a final concentration of 3μM and then 25 DAPI-positive nuclei were sorted into each well of a plate with 20μl EB buffer. '''was at 11 uL, added 11 uL gHCL''' '''added 39.0 uL of beads, and eluted in 8 uL''' 7. Bring each well to 10 uL with N-H2O. Add 10 uL 8M guanidine HCl for a total of 20 uL. **For SPRI beads add 1.8X (36 uL) beads per well and follow beads purification protocol **ELUTE IN 8, leave beads in solution 8. Fill in reaction '''2 uL taq 5X per reaction used''' *Add 1.5 uL of 5X taq polymerase, mix thoroughly. Run at 72C for 3 minutes. 9. HiScribe T7 (NEB) T7 Protocol, IVT '''added hiscribe mastermix directly into tubes after taq5x incubation''' HiScribe (NEB) T7 protocol, IVT 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!!!! (10) uL DNA template (list 1 ug) 2 uL 10X Transcription Buffer 2 uL 100 mM ATP 2 uL 100 mM CTP 2 uL 100 mM GTP 2 uL 100 mM UTP 2 uL T7 Enzyme Mix _______________________ 22 uL total c. Incubate reactions at 37C overnight for ~16-19 hours. 10. Clean with ZR-96 RNA Clean & Concentrator, put solution and all beads on columns. *using beads could be an option? *elute samples in 9.0 uL of N-H2O *quanitate/view on TBU gel, only 1 sample? '''AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO''' *Amounts of RNA and water to add for MMLV RT (50 ng RNA) {| {{table}} | align="center" style="background:#f0f0f0;"|'''10/7/2015''' | align="center" style="background:#f0f0f0;"|'''intensity''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||1763.757444||5|||||||| |- | ||895.2045194||2.5||in 10 uL||||for 50 ng||H2O |- | 1. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep1||33570.21118||96.55013139||965.5013139||||0.517865686||8.482134314 |- | 2. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep2||15673.5593||45.03728923||450.3728923||||1.110191152||7.889808848 |- | 3. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep3||25727.79598||73.97690936||739.7690936||||0.675886576||8.324113424 |- | 4. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep1||28224.6736||81.16379904||811.6379904||||0.616038192||8.383961808 |- | 5. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep2||36786.24018||105.806991||1058.06991||||0.472558567||8.527441433 |- | 6. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep3||35388.89339||101.7849367||1017.849367||||0.491231823||8.508768177 |- | 7. T7-nXTv2_i5-1, 5X tn5059, 6ng DNA, buffer D6||108457.5135||312.1020574||3121.020574||/4||0.640816026||8.359183974 |- | 8. T7-nXTv2_i5-1, 5X tn5059, NTC, buffer D6||189.5401283||0.468850339||4.688503394||1||||9 |- | |} 11. single strand synthesis MMLV RT (Clontech) *Followed protocol for [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/SMART_MMLV_RT SMART MMLV Reverse Transcriptase] a. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 11.5 uL with Nuclease free H2O b. heat the mixture to 70C for 3 minutes. Immediately cool on ice. c. Add the following to the reaction. 4 uL 5X first strand buffer 2 uL dNTP mix 2 uL 100 mM DTT .5 uL SMART MMLV RT and mix (ADD LAST!!!!!) ___________________________ 20 uL total d. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes. e. Terminate the reaction by heating at 70C for 10 minutes 12. RNase H digestion *Use .5 Units for 20 uL reaction. Stock is at 5 Units/ul, thus dilute 10X and use 1 uL per reaction. a. Add 1 uL of 10X diluted Rnase H to the reaction. b. Incubate for 20 minutes at 37C. 13. Second strand synthesis: Adding P-sss_nXTv2 primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since P_sss_nXTv2 has a high Tm. a. Add 2.5 uL of 20 uM P-sss_nXTv2 to each reaction. b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice. c. Add 5.875 uL taq5X to reactions, Incubate at 72C for 8 minutes. 14. Clean DNA with AMPURE beads depending. *'''USED AMPURE DNA BEADS AT 1:1.8X RATIO''' *add 54 uL beads per reaction *'''ADDED 7 uL N-H20 TO BEADS''' *'''LEFT BEADS IN SOLUTION, DID NOT REMOVE BEADS FROM ADDED N-H20''' 15. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6 '''RAN TAGMENTATION ON BEADS''' 2 uL custom tagmentation buffer '''USED BUFFER TA, 10% FINAL DMF''' X uL cDNA sample X uL Nuclase free H2O 2 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST) ___________ 11 uL total solution Incubate at 55C for 6 minutes, cool briefly on ice after 16. Add 4 uL N-H2O, then added 15 uL 8M guanidine HCl 17. Purified on DNA clean and concentrator, 5 volumes DNA binding buffer, elute in 13 uL N-H2O 18. PCR addition of barcodes '''USING NEXTERA XT V2 modified i5 (T7-1_S5XX) AND i7 (N7XX) SEQUENCES!!!!!''' Samples: 1. i502_T7-1_short-i701_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep1 2. i502_T7-1_short-i702_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep2 3. i502_T7-1_short-i703_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep3 4. i502_T7-1_short-i704_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep1 5. i503_T7-1_short-i701_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep2 6. i503_T7-1_short-i702_long, T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep3 7. i503_T7-1_short-i703_long, T7-nXTv2_i5-1, 5X tn5059, 6ng DNA, buffer D6 8. i503_T7-1_short-i704_long, T7-nXTv2_i5-1, 5X tn5059, NTC, buffer D6 KAPA SYBR FAST qPCR mix until saturation, X35 cycles 17 uL 2X kapa sybr fast 2 uL scT7_S5XX index primer, 2 uL F, (i5XX_T7-1_long or short) '''USED XXX''' 2 uL nXTv2_i7XX index primer, 2 uL R, (i7XX_long or i7XX_orig) 13 uL DNA template (use half RT reaction) _____________ 34 uL KAPA SYBR cycles: 72C 3m, 95C 30s, (95C for 10s, 63C for 30s, 72C for 1 min) X15, 72C for 3 min, 4C forever *terminate before curves saturate (usually cycle 6-8) 19. Pool all samples run on 6% PAGE gel, Qubit. Gel Size selection *gel size select from 220-1000 bp, follow gel size selection protocol *do not need to include controls. ===Results=== *total RNA. {| {{table}} | align="center" style="background:#f0f0f0;"|'''10/7/2015''' | align="center" style="background:#f0f0f0;"|'''intensity''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||1763.757444||5|||||||| |- | ||895.2045194||2.5||in 10 uL||||for 50 ng||H2O |- | 1. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep1||33570.21118||96.55013139||965.5013139||||0.517865686||8.482134314 |- | 2. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep2||15673.5593||45.03728923||450.3728923||||1.110191152||7.889808848 |- | 3. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep3||25727.79598||73.97690936||739.7690936||||0.675886576||8.324113424 |- | 4. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep1||28224.6736||81.16379904||811.6379904||||0.616038192||8.383961808 |- | 5. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep2||36786.24018||105.806991||1058.06991||||0.472558567||8.527441433 |- | 6. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep3||35388.89339||101.7849367||1017.849367||||0.491231823||8.508768177 |- | 7. T7-nXTv2_i5-1, 5X tn5059, 6ng DNA, buffer D6||108457.5135||312.1020574||3121.020574||/4||0.640816026||8.359183974 |- | 8. T7-nXTv2_i5-1, 5X tn5059, NTC, buffer D6||189.5401283||0.468850339||4.688503394||1||||9 |- | |} *TBU gel after IVT, Guanidine HCl to remove proteins [[File:ZhangLab 2 2015-10-05 15hr 05min-labeled.jpg|600px]] *qPCR curves [[File:2015-10-07 hiscribe TA v D6 comparison.bmp|600px]] *TBE after PCR amplification [[File:ZhangLab 2 2015-10-07 16hr 32min-labeled.jpg|600px]] *gel size selection amounts {| {{table}} | align="center" style="background:#f0f0f0;"|'''10/7/2015 gel size selection''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||5344.98624||5|||| |- | ||2976.702589||2.5||for 50 ng||X2 |- | 1. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep1||21574.08855||22.13171298||2.259201538||4.518403075 |- | 2. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep2||25469.0217||26.24326983||1.905250387||3.810500774 |- | 3. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer D6 Rep3||14060.63573||14.20038598||3.521031052||7.042062104 |- | 4. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep1||12155.62807||12.1894279||4.101915233||8.203830466 |- | 5. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep2||10532.47809||10.47600352||4.772812446||9.545624893 |- | 6. T7-nXTv2_i5-1, 5X tn5059, 539 cells, buffer TA Rep3||9135.697926||9.001538924||5.554605765||11.10921153 |- | 7. T7-nXTv2_i5-1, 5X tn5059, 6ng DNA, buffer D6||24780.47545||25.51642884||0||0 |- | 8. T7-nXTv2_i5-1, 5X tn5059, NTC, buffer D6||-143.1827782||-0.793403394||1||2 |- | |||||||| |- | |} *gel size selection [[File:ZhangLab 2 2015-10-07 18hr 09min-labeled.jpg|600px]] *gel size selection validation *ran on TBU cuz no TBEs were available. looks fine. [[File:ZhangLab 2 2015-10-08 11hr 17min-labeled.jpg|600px]] ===conclusions=== *hard to tell which is better with limited sequencing data, regardless, viewing on ucsc tracts cannot tell if one gives better data or not. both look worse since not getting as many peaks as before. *possibilities for getting not as good data are: **3 uL cells in 1 uL lysis buffer, EDTA stopping **size select 300 and up **used maxiscript not hiscribe **new RNA processing after 2x double stranded synthesis **data not as good as when comparing to nXTV2 validation [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-8-3 Update of THS-seq protocol sequences to use Nextera XT V2 sequences] **'''cell health? very high possibility since this has been seen before when replicating ATAC-seq, maybe cells are in quiescent state. peaks present are well defined, which gives more evidence to this''' [[File:D6 versus TA buffer ucsc tract.png|600px]]
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