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==Validating changes made to THS-seq protocol for combinatorial sequencing== *small changes in amounts of reagents used were made in: [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-9-21 multiplex combinatorial cellular indexing adaptation to THS-seq using nXTv2 sequence detail and protocol], testing these small changes to ensure ample amounts of IVT RNA are generated, and libraries are generated successfully after PCR. *questions answered: **Does tagmentation work fine in the presence of protease inhibitors, and in 6.4 uL tagmentation reaction? **comparable IVT yields when using 3 uL cells, 1 uL 4X lysis buffer? **stopping reation with EDTA 15m at 37C fine? **pooling and redistributing result in loss? **using 1/3 beads (36 uL v 54 uL) fine? **eluting in 6 uL off of SPRI beads to hard? **1.5 uL taq5X versus 2.4 uL? **~1/2 IVT reagents work fine (8 v 4.8 uL)? **after 2x stranded cDNA synthesis, no water addition, only 1.8X beads work fine? **SPRI afer 2X stranded cDNA synthesis eluted in 10 uL? (run beads on gel and see if any product still stuck?) **second tagmentation in 16 uL? **adding 4 uL protease to 16 uL? **adding 5 uL 5X taq for end fill in? **PCR with addition only of primers and 1 uL taq5X (same for SYBR green) *questions not answered: **Using 16% DMF final concentration in solution, any effect on dissociating nuclei? didn't with 10% **sorting by FACS with DAPI added necessary? just add by dilution? **96 well RNA purification plate for all samples, versus doing beads? *went through scheme #1 in this post, PCR didnt work since to much DMF in reaction for PCR to work. [[File:2015-10-02 experiemnts summary.png|600px]] *reagents etc needed: **taq5X ~ 1 mL (100) **clontech MMLV, 1/2 kit, and 1 order of dNTPS (125) **SPRI beads (~10 mL per run) (175) **maxiscript ~1/2 kit per run (150) **zymo ZR RNA clean and concentrator plate 1 plate, (200) **qiagen protease, 1/2 (70) ===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_long-i701_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep1 2. i502_T7-1_long-i702_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep2 3. i502_T7-1_long-i703_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep3 4. i503_T7-1_long-i701_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep4 5. i503_T7-1_long-i702_long, T7-nXTv2_i5-1, 5X tn5059, 6 ng pure DNA 6. i503_T7-1_long-i703_long, T7-nXTv2_i5-1, 5X tn5059, NTC ===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 '''each reaction had 333 cells in each tube''' '''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 1 volume (6.5 uL) 40 mM EDTA. Incubate for 15 mins at 37C. 6. Nuclei pooling and recovery '''just redistributed the same nuclei to tubes''' 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 13 uL, added 8 uL gHCL''' 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 6, leave beads in solution''' 8. Fill in reaction *Add 1.5 uL of 5X taq polymerase, mix thoroughly. Run at 72C for 3 minutes. 9. 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!!!! X uL DNA template (list 1 ug) 1.2 uL 10X Transcription Buffer 0.6 uL 10 mM ATP 0.6 uL 10 mM CTP 0.6 uL 10 mM GTP 0.6 uL 10 mM UTP 1.2 uL T7 Enzyme Mix _______________________ 12.3 uL total b. 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;"|'''9/25/2015''' | 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;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||1540.165708||5|||||||||| |- | ||441.3047926||2.5||in 10 ul||for 50 ng|||||| |- | 1. i502_T7-1_long-i701_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep1||3737.306598||9.998678313||99.98678313||5.000660931|||||| |- | 2. i502_T7-1_long-i702_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep2||3910.542279||10.3928039||103.928039||4.811021211|||||| |- | 3. i502_T7-1_long-i703_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep3||10229.91571||24.76990419||247.6990419||2.01857866|||||| |- | 4. i503_T7-1_long-i701_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep4||4889.326632||12.61961973||126.1961973||3.962084521|||||| |- | 5. i503_T7-1_long-i702_long, T7-nXTv2_i5-1, 5X tn5059, 6 ng pure DNA||97614.73967||223.5776485||2235.776485||0.223635951||/4||55.89441214||0.894543803 |- | 6. i503_T7-1_long-i703_long, T7-nXTv2_i5-1, 5X tn5059, NTC||257.4794671||2.081782092||20.81782092||1 uL|||||| |- | ||0||1.495994883||14.95994883||0|||||| |- | ||-83.77946196||1.305389637||13.05389637||38.30274009|||||| |- | ||||||||#DIV/0!|||||| |- | |} 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 sss_nXTv2 primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_nXTv2 has a high Tm. a. Add 2.5 uL of 20 uM 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. elute in 10 uL *'''USED AMPURE DNA BEADS AT 1:1.8X RATIO''' *add 54 uL beads per reaction *elute on 10 uL 15. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6 3 uL custom tagmentation buffer X uL cDNA sample X uL Nuclase free H2O 3 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST) ___________ 16 uL total solution Incubate at 55C for 6 minutes, cool briefly on ice after 16. Protease digestion of transposase, protease inactivation To each tube, add: 4 uL Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final []) Incubate: 50C 10 minutes, 70C 20 minutes 17. Fill in reaction *Add 5 uL 5X taq polymerase, run at 72C for 3 minutes. 18. PCR addition of barcodes '''USING NEXTERA XT V2 modified i5 (T7-1_S5XX) AND i7 (N7XX) SEQUENCES!!!!!''' Samples: 1. i502_T7-1_long-i701_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep1 2. i502_T7-1_long-i702_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep2 3. i502_T7-1_long-i703_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep3 4. i503_T7-1_long-i701_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep4 5. i503_T7-1_long-i702_long, T7-nXTv2_i5-1, 5X tn5059, 6 ng pure DNA 6. i503_T7-1_long-i703_long, T7-nXTv2_i5-1, 5X tn5059, NTC KAPA SYBR FAST qPCR mix until saturation, X35 cycles 1 uL Taq5X 2 uL scT7_S5XX index primer, 2 uL F, (i5XX_T7-1_long) '''USED XXX''' 2 uL nXTv2_i7XX index primer, 2 uL R, (i7XX_long or i7XX_orig) 1 uL 25X SYBR green 25 uL DNA template (use half RT reaction) _____________ 30 uL KAPA SYBR cycles: 72C 3m, 95C 30s, (95C for 10s, 63C for 30s, 72C for 1:30 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;"|'''9/25/2015''' | 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;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||1540.165708||5|||||||||| |- | ||441.3047926||2.5||in 10 ul||for 50 ng|||||| |- | 1. i502_T7-1_long-i701_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep1||3737.306598||9.998678313||99.98678313||5.000660931|||||| |- | 2. i502_T7-1_long-i702_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep2||3910.542279||10.3928039||103.928039||4.811021211|||||| |- | 3. i502_T7-1_long-i703_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep3||10229.91571||24.76990419||247.6990419||2.01857866|||||| |- | 4. i503_T7-1_long-i701_long, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep4||4889.326632||12.61961973||126.1961973||3.962084521|||||| |- | 5. i503_T7-1_long-i702_long, T7-nXTv2_i5-1, 5X tn5059, 6 ng pure DNA||97614.73967||223.5776485||2235.776485||0.223635951||/4||55.89441214||0.894543803 |- | 6. i503_T7-1_long-i703_long, T7-nXTv2_i5-1, 5X tn5059, NTC||257.4794671||2.081782092||20.81782092||1 uL|||||| |- | ||0||1.495994883||14.95994883||0|||||| |- | ||-83.77946196||1.305389637||13.05389637||38.30274009|||||| |- | ||||||||#DIV/0!|||||| |- | |} *TBU gel after IVT, Guanidine HCl to remove proteins *looks like it worked fine with 333 cells [[File:ZhangLab 2 2015-09-25 13hr 32min-labeled.jpg|600px]] *qPCR curves *curves didn't go up, something wrong? (was to much DMF in PCR reaction) [[File:2015-09-25 THS minor protocl changes validation.png|600px]] *TBE after PCR amplification *can tell that fragmentation as occured, after comparing to gel run of what is left over on beads [[File:ZhangLab 2 2015-09-25 20hr 33min-labeled.jpg|600px]] *running of left over beads after elution on TBE *can tell DNA is still intact at this point [[File:ZhangLab 2 2015-09-25 20hr 13min-labeled.jpg|600px]] *'''9/28/2015 UPDATE''' *cleaned with zymo clean and concentrator and PCR worked very well. DNA fragments were present in solution, just DMF did not allow PCR to run correctly. after cleaning and readdin primers, worked fine. *also used KAPA SYBR fast, and 50 uL reaction volume *will try different methods of RNA processing to see which gives best smears [[File:ZhangLab 2 2015-09-28 15hr 11min-labeled.jpg|600px]] *gel size selection amounts *gel size selection *gel size selection validation ===conclusions=== *submitted for sequencing
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