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===Protocol=== '''AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO''' Samples: 1. 9/24/2015 RNA #1+2, i502_T7-1_short-i701_orig, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep1,2 2. 9/24/2015 RNA #3, i502_T7-1_short-i702_orig, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep3 3. 9/24/2015 RNA #5, i502_T7-1_short-i703_orig, T7-nXTv2_i5-1, 5X tn5059, 6 ng pure DNA 4. 9/24/2015 RNA #6, i503_T7-1_short-i701_orig, T7-nXTv2_i5-1, 5X tn5059, NTC *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. Exo I treatment, 3 uL (1 uL per 10 uL of reaction (20 units/uL), incubate 37C for 20 minutes 15. Clean with Zymo DNA clean and concentrator, 5 volumes DNA binding solution, elute 6 uL 16. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6 2 uL custom tagmentation buffer '''USED 50% DMF, 10% FINAL IN REACTION''' X uL cDNA sample X uL Nuclase free H2O 2 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST) ___________ 10 uL total solution Incubate at 55C for 6 minutes, cool briefly on ice after 17. Protease digestion of transposase, protease inactivation To each tube, add: 2 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 18. PCR addition of barcodes '''USING NEXTERA XT V2 modified i5 (T7-1_S5XX) AND i7 (N7XX) SEQUENCES!!!!!''' Samples: 1. 9/24/2015 RNA #1+2, i502_T7-1_short-i701_orig, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep1,2 2. 9/24/2015 RNA #3, i502_T7-1_short-i702_orig, T7-nXTv2_i5-1, 5X tn5059, 500 cells Rep3 3. 9/24/2015 RNA #5, i502_T7-1_short-i703_orig, T7-nXTv2_i5-1, 5X tn5059, 6 ng pure DNA 4. 9/24/2015 RNA #6, i503_T7-1_short-i701_orig, T7-nXTv2_i5-1, 5X tn5059, NTC KAPA SYBR FAST qPCR mix until saturation, X35 cycles 25 uL kapa sybr fast 4 uL scT7_S5XX index primer, 2 uL F, (i5XX_T7-1_long) '''USED XXX''' 4 uL nXTv2_i7XX index primer, 2 uL R, (i7XX_long or i7XX_orig) 5 uL N-H2O 12 uL DNA template (use half RT reaction) _____________ 50 uL KAPA SYBR cycles: 72C 3m, 95C 30s, (95C for 10s, 59C 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.
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