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Arichard:Protocols/CEL Seq
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==CEL-Seq protocol validation as of 2/28/2013== * Protocol modified to use common sequence primer with Illumina 5' Adapter and T7 promoter (CS-5primeAD-T7). * 100 ng/ul total RNA will be used as input (from Rui). * I will work with 16 tubes at a time. * For one set of 16 tubes, I want 2 x 1 ng rxns (+ control), 12 x 100 pg rxns (experimental), and 2 x no RNA (no template controls, NTC). * For the second set of 16, I want 2 x 100 pg rxns (+ control), 12 x 10 pg rxns (experimental, approx. single cell amount), and 2 x no RNA (NTCs). * Keep all RNA on ice at all times. * Work in the hood. EtOH, RNase away, and UV treat all surfaces and tools. UV treat water for dilutions. * Use low-bind tubes. ===Prep=== * RT primers must be diluted to appropriate concentrations depending on the input: ** For 1 ng of total RNA, use 0.5 ul of 32 pmol/ul. ** For 100 pg of total RNA, use 0.5 ul of 3.2 pmol/ul. ** For 10 pg of total RNA, use 0.5 ul of 0.32 pmol/ul. * See diluted RT primer layout under Arichard:Reagents. ====Dilute total RNA to 2 ng/ul, on ice==== # Add 1 ul 100 ng/ul RNA to 9 ul H2O --> 10 ul 10 ng/ul RNA. # Add 2 ul 10 ng/ul RNA to 8 ul of H2O --> 10 ul 2 ng/ul RNA. * Store all at -80 degC. ====Dilute ERCC RNA Spike-in Control to 1:10,000, on ice==== # Add 1 ul of undiluted ERCC to 9 ul H2O --> 10 ul 1:10 ERCC. # Add 1 ul of 1:10 ERCC to 9 ul H2O --> 10 ul 1:100 ERCC. # Add 1 ul of 1:100 ERCC to 9 ul H2O --> 10 ul 1:1000 ERCC. # Add 1 ul of 1:1000 ERCC to 9 ul H2O --> 10 ul 1:10000 ERCC. * Store all at -80 degC. ====Mix total RNA and ERCC RNA Spike-in Control==== # Add 5 ul 2 ng/ul RNA to 2 ul 1:10000 ERCC --> 7 ul 1.42 ng/ul RNA 1:35000 ERCC. ## Add 0.7 ul of this to 0.5 ul 32 pmol/ul primer for 1 ng total RNA samples. ## Need total of 2 x 0.7 ul + 2 ul for dilution to 142 pg/ul RNA 1:350000 ERCC = 3.4 ul. # Add 2 ul 1.42 ng/ul RNA 1:35000 ERCC to 18 ul H2O --> 20 ul 142 pg/ul RNA 1:350000 ERCC. ## Add 0.7 ul of this to 0.5 ul 3.2 pmol/ul primer for 100 pg total RNA samples. ## Need 14 x 0.7 ul + 1 ul for dilution to 14.2 pg/ul RNA 1:3500000 ERCC = 10.8 ul. # Add 1 ul 142 pg/ul RNA 1:350000 ERCC to 9 ul H2O --> 10 ul 14.2 pg/ul RNA 1:3500000 ERCC. ## Add 0.7 ul of this to 0.5 ul 0.32 pmol/ul primer for 10 pg samples. ## Need 12 x 0.7 ul = 8.4 ul. ===Protocol=== ====RT reaction, first strand==== # On ice block, add 0.5 ul of each RT primer at appropriate concentration (see prep above) to each tube. # Add 0.7 ul of each sample/ERCC mixture to respective tubes. # Flick and spin tubes to collect, then return to ice block. # Incubate 10 min at 70 degC to anneal RT primers. # Add 0.8 ul of RT mix per rxn: ## 0.2 ul 10X first strand buffer. ## 0.4 ul dNTP mix. ## 0.1 ul RNase inhibitor. ## 0.1 ul ArrayScript. # Flick and spin tubes to collect, then return to ice block. # Incubate 2 hrs @ 42 degC for RT rxn. ====Second strand synthesis==== # Move tubes to ice block and add 8 ul of second strand mix per rxn: ##6.3 ul DDW H2O. ## 1 ul Second Strand buffer. ## 0.4 ul dNTP mix. ## 0.2 ul DNA Polymerase ## 0.1 ul RNaseH. # Flick and spin to collect, then incubate 2 hrs @ 16 degC. # Pool samples and add to UV treated EtOH precipitation mix (for 16 samples, 10 ul/samples): ## 400 ul 100% EtOH. ## 32 ul 3M NaAc. ## 1 ul glycoblue. # Incubate at least 1 hr @ -80 degC. # Spin 30 min @ 4 degC. # Remove supernatant and wash with 750 ul 75% EtOH. # Spin again 10 min @ 4 degC. # Remove supernatant and let dry 5 min. # Resuspend in 4.76 ul UV treated H2O. * Stopping point: Store @ -20 degC. ====Common primer==== # On ice, add 0.64 ul 10X NEB Buffer 2, 0.5 ul of 10 pmol/ul CS-5primeAD-T7 primer to 4.76 ul of ds cDNA. Incubate this for 2 min @ 95 degC, then 1 min @ 66 degC. Tm for 50% is 69 degC, and 66 degC gives 90%. # Add 0.5 ul Bst large fragment and incubate for 30 min @ 65 degC, followed by 20 min @ 80 degC to heat inactivate. ====IVT==== # On ice, add 9.6 ul of IVT mix per rxn: ## 1.6 ul ATP. ## 1.6 ul CTP. ## 1.6 ul GTP. ## 1.6 ul UTP. ## 1.6 ul 10X T7 buffer. ## 1.6 ul T7 enzyme. # Flick and spin to collect. Incubate 13 hrs @ 37 degC, followed by 4 degC forever. ====Mg2+ fragmentation==== # On ice, add 6.25 of 10X NEB fragmentation buffer to 25 ul of sample. # Incubate 3 min @ 94 degC. # Immediately move to ice and add 3.13 ul fragmentation stop buffer. # Add 70 RNA binding buffer, then 100 ul 100% EtOH. Add to zymo column and spin 1 min @ 12,000 g. # Discard flowthrough, add 200 ul RNA prep buffer and spin. # Discard flowthrough, add 400 ul RNA wash buffer and spin 30 sec. # Discard flowthrough and spin 2 min in the same tube. # Transfer column to RNase free tube and add 10 ul of RNase free H2O @ RT to elute. Let stand 1 min, then spin 1 min @ 10,000 g. # Repeat elution. Total volume = 20 ul. # Run bioanalyzer.
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