Arichard:Protocols/CEL Seq: Difference between revisions
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>Andrew (Created page with "==CEL-Seq protocol validation== * Protocol modified to use common sequence primer with Illumina 5' Adapter and T7 promoter (CS-5primeAD-T7). * 100 ng/ul total RNA will be use...") |
>Andrew No edit summary |
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* 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 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). | * 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=== | |||
* Sample layout is as follows: | * Sample layout is as follows: | ||
Line 37: | Line 43: | ||
|} | |} | ||
=== | * Barcoded RT primers are in 48 wells of a 92 well plate at 200 uM each, in the following layout: | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Barcode number''' | |||
| align="center" style="background:#f0f0f0;"|'''1''' | |||
| align="center" style="background:#f0f0f0;"|'''2''' | |||
| align="center" style="background:#f0f0f0;"|'''3''' | |||
| align="center" style="background:#f0f0f0;"|'''4''' | |||
| align="center" style="background:#f0f0f0;"|'''5''' | |||
| align="center" style="background:#f0f0f0;"|'''6''' | |||
| align="center" style="background:#f0f0f0;"|'''7''' | |||
| align="center" style="background:#f0f0f0;"|'''8''' | |||
| align="center" style="background:#f0f0f0;"|'''9''' | |||
| align="center" style="background:#f0f0f0;"|'''10''' | |||
| align="center" style="background:#f0f0f0;"|'''11''' | |||
| align="center" style="background:#f0f0f0;"|'''12''' | |||
|- | |||
| A||1||2||3||4||5||6||7||8||9||10||11||12 | |||
|- | |||
| B||13||14||15||16||17||18||19||20||21||22||23||24 | |||
|- | |||
| C||25||26||27||28||29||30||31||32||33||34||35||36 | |||
|- | |||
| D||37||38||39||40||41||42||43||44||45||46||47||48 | |||
|- | |||
| E||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty | |||
|- | |||
| F||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty | |||
|- | |||
| G||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty | |||
|- | |||
| H||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty||Empty | |||
|- | |||
| | |||
|} | |||
* Serial dilutions will be necessary to dilute the RT primers to the correct concentration. | |||
====Dilute primers to appropriate concentrations==== | ====Dilute RT primers to appropriate concentrations==== | ||
# | # Dilute primers to 32 pmol/ul, 3.2 pmol/ul, and 0.32 pmol/ul, depending on input (Note that each primer must be diluted separately for each sample): | ||
## Add 16 ul of 100 pmol/ul primer to 84 ul H2O --> 100 ul 32 pmol/ul primer. Use 0.5 ul of this per 1 ng total RNA. | |||
## Add 10 ul of 32 pmol/ul primer to 90 ul H2O --> 100 ul 3.2 pmol/ul primer. Use 0.5 ul of this per 100 pg total RNA. | |||
## Add 10 ul of 3.2 pmol/ul primer to 90 ul H2O --> 100 ul 0.32 pmol/ul primer. Use 0.5 ul of this per 10 pg total RNA. | |||
# 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. |
Revision as of 00:44, 8 February 2013
CEL-Seq protocol validation
- 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
- Sample layout is as follows:
Sample # | RNA input | Sample # | RNA input | Sample # | RNA input | Sample # | RNA input |
1 | 1 ng | 9 | 100 pg | 17 | 100 pg | 25 | 10 pg |
2 | 1 ng | 10 | 100 pg | 18 | 100 pg | 26 | 10 pg |
3 | 100 pg | 11 | 100 pg | 19 | 10 pg | 27 | 10 pg |
4 | 100 pg | 12 | 100 pg | 20 | 10 pg | 28 | 10 pg |
5 | 100 pg | 13 | 100 pg | 21 | 10 pg | 29 | 10 pg |
6 | 100 pg | 14 | 100 pg | 22 | 10 pg | 30 | 10 pg |
7 | 100 pg | 15 | NTC | 23 | 10 pg | 31 | NTC |
8 | 100 pg | 16 | NTC | 24 | 10 pg | 32 | NTC |
- Barcoded RT primers are in 48 wells of a 92 well plate at 200 uM each, in the following layout:
Barcode number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
A | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
B | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
C | 25 | 26 | 27 | 28 | 29 | 30 | 31 | 32 | 33 | 34 | 35 | 36 |
D | 37 | 38 | 39 | 40 | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 |
E | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty |
F | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty |
G | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty |
H | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty | Empty |
- Serial dilutions will be necessary to dilute the RT primers to the correct concentration.
Dilute RT primers to appropriate concentrations
- Dilute primers to 32 pmol/ul, 3.2 pmol/ul, and 0.32 pmol/ul, depending on input (Note that each primer must be diluted separately for each sample):
- Add 16 ul of 100 pmol/ul primer to 84 ul H2O --> 100 ul 32 pmol/ul primer. Use 0.5 ul of this per 1 ng total RNA.
- Add 10 ul of 32 pmol/ul primer to 90 ul H2O --> 100 ul 3.2 pmol/ul primer. Use 0.5 ul of this per 100 pg total RNA.
- Add 10 ul of 3.2 pmol/ul primer to 90 ul H2O --> 100 ul 0.32 pmol/ul primer. Use 0.5 ul of this per 10 pg total RNA.
- 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.