Arichard:Protocols/CEL Seq: Difference between revisions

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* 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
|-
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|}
* Serial dilutions will be necessary to dilute the RT primers to the correct concentration.


====Dilute RT primers to appropriate concentrations====
====Dilute RT primers to appropriate concentrations====
Line 87: Line 49:
## 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 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.
## 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.
====Dilute total RNA to 2 ng/ul, on ice====
# Dilute ERCC RNA Spike-in Control to 1:10,000, on ice:
# Add 1 ul 100 ng/ul RNA to 9 ul H2O --> 10 ul 10 ng/ul RNA.
## Add 1 ul of undiluted ERCC to 9 ul H2O --> 10 ul 1:10 ERCC.
# Add 2 ul 10 ng/ul RNA to 8 ul of H2O --> 10 ul 2 ng/ul RNA.
## 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.
====Dilute ERCC RNA Spike-in Control to 1:10,000, on ice====
## Add 1 ul of 1:1000 ERCC to 9 ul H2O --> 10 ul 1:10000 ERCC.
# 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.
 
===Protocol===
 
====RT reaction, first strand====
 
# 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 74:
## 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.
to be continued

Revision as of 17:19, 14 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

Dilute RT primers to appropriate concentrations

  1. 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):
    1. 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.
    2. 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.
    3. 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

  1. Add 1 ul 100 ng/ul RNA to 9 ul H2O --> 10 ul 10 ng/ul RNA.
  2. Add 2 ul 10 ng/ul RNA to 8 ul of H2O --> 10 ul 2 ng/ul RNA.

Dilute ERCC RNA Spike-in Control to 1:10,000, on ice

  1. Add 1 ul of undiluted ERCC to 9 ul H2O --> 10 ul 1:10 ERCC.
  2. Add 1 ul of 1:10 ERCC to 9 ul H2O --> 10 ul 1:100 ERCC.
  3. Add 1 ul of 1:100 ERCC to 9 ul H2O --> 10 ul 1:1000 ERCC.
  4. Add 1 ul of 1:1000 ERCC to 9 ul H2O --> 10 ul 1:10000 ERCC.

Protocol

RT reaction, first strand

  1. Add 5 ul 2 ng/ul RNA to 2 ul 1:10000 ERCC --> 7 ul 1.42 ng/ul RNA 1:35000 ERCC.
    1. Add 0.7 ul of this to 0.5 ul 32 pmol/ul primer for 1 ng total RNA samples.
    2. Need total of 2 x 0.7 ul + 2 ul for dilution to 142 pg/ul RNA 1:350000 ERCC = 3.4 ul.
  2. Add 2 ul 1.42 ng/ul RNA 1:35000 ERCC to 18 ul H2O --> 20 ul 142 pg/ul RNA 1:350000 ERCC.
    1. Add 0.7 ul of this to 0.5 ul 3.2 pmol/ul primer for 100 pg total RNA samples.
    2. Need 14 x 0.7 ul + 1 ul for dilution to 14.2 pg/ul RNA 1:3500000 ERCC = 10.8 ul.
  3. Add 1 ul 142 pg/ul RNA 1:350000 ERCC to 9 ul H2O --> 10 ul 14.2 pg/ul RNA 1:3500000 ERCC.
    1. Add 0.7 ul of this to 0.5 ul 0.32 pmol/ul primer for 10 pg samples.
    2. Need 12 x 0.7 ul = 8.4 ul.

to be continued