Arichard:Protocols/CEL Seq: Difference between revisions
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==CEL-Seq protocol validation== | ==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). | * Protocol modified to use common sequence primer with Illumina 5' Adapter and T7 promoter (CS-5primeAD-T7). | ||
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===Prep=== | ===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 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. | ## Add 0.7 ul of this to 0.5 ul 32 pmol/ul primer for 1 ng total RNA samples. | ||
Line 104: | Line 43: | ||
## Add 0.7 ul of this to 0.5 ul 0.32 pmol/ul primer for 10 pg samples. | ## 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. | ## 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. |
Latest revision as of 21:19, 28 February 2013
CEL-Seq protocol validation as of 2/28/2013[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
RT reaction, first strand[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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.