Daniel:Protocols/RNASort: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 61: | Line 61: | ||
**0.5% SDS (v/v) | **0.5% SDS (v/v) | ||
**500 ug/mL proteinase K | **500 ug/mL proteinase K | ||
==Buffer Prep== | |||
===RPHB=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#D8E4BC;font-size:12pt" align="center" | |||
| width="112" height="37" | Reagent | |||
| width="78" | Stock Concentration | |||
| width="77" | Final Concentration | |||
| width="65" | Dilution | |||
| width="127" | Volume in 10 mL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | SSC Buffer | |||
| align="center" valign="bottom" | 20X | |||
| align="center" valign="bottom" | 2X | |||
| align="center" align="center" valign="bottom" | 1:10 | |||
| align="center" valign="bottom" | 1 mL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Ammonium Sulfate | |||
| align="center" valign="bottom" | NA, powder | |||
| align="center" valign="bottom" | 2.1 M | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | 2.775g | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | EDTA | |||
| align="center" valign="bottom" | 0.5 M | |||
| align="center" valign="bottom" | 10 mM | |||
| align="center" align="center" valign="bottom" | 1:50 | |||
| align="center" valign="bottom" | 200 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | E. coli tRNA | |||
| align="center" valign="bottom" | 20 mg/mL | |||
| align="center" valign="bottom" | 1 mg/mL | |||
| align="center" align="center" valign="bottom" | 1:20 | |||
| align="center" valign="bottom" | 500 uL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | BSA | |||
| align="center" valign="bottom" | 50 mg/mL | |||
| align="center" valign="bottom" | 500 ug/mL | |||
| align="center" valign="bottom" | 1:100 | |||
| align="center" valign="bottom" | 100 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Formamide | |||
| align="center" align="center" valign="bottom" | 100% | |||
| align="center" align="center" valign="bottom" | 25% | |||
| align="center" align="center" valign="bottom" | 1:04 | |||
| align="center" valign="bottom" | 2.5 mL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" valign="bottom" | - | |||
| align="center" valign="bottom" | - | |||
| align="center" valign="bottom" | - | |||
| align="center" valign="bottom" | Fill to 10mL (~5 mL) | |||
|} | |||
===Sorting Buffer=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#D8E4BC;font-size:12pt" align="center" | |||
| width="112" height="30" | Reagent | |||
| width="78" | Stock Concentration | |||
| width="77" | Final Concentration | |||
| width="65" | Dilution | |||
| width="127" | Volume in 10 mL | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | SSC Buffer | |||
| 20X | |||
| 2X | |||
| align="center" | 1:10 | |||
| 1 mL | |||
|- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | |||
| height="15" | Ammonium Sulfate | |||
| NA, powder | |||
| 1.5 M | |||
| NA | |||
| 1.982 g | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | EDTA | |||
| 0.5 M | |||
| 5 mM | |||
| 1:100 | |||
| 100 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | |||
| height="15" | nfH2O | |||
| - | |||
| - | |||
| - | |||
| Fill to 10mL (~9 mL) | |||
|} | |||
===Reverse Cross-Linking Buffer=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#D8E4BC;font-size:12pt" align="center" | |||
| width="112" height="30" | Reagent | |||
| width="78" | Stock Concentration | |||
| width="77" | Final Concentration | |||
| width="65" | Dilution | |||
| width="127" | Volume in 10 mL | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | NaCl | |||
| NA, powder | |||
| 100 mM | |||
| - | |||
| 58.4 mg | |||
|- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | |||
| height="15" | Tris | |||
| 1 M | |||
| 10 mM | |||
| 1:100 | |||
| 100 uL | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | EDTA | |||
| 0.5 M | |||
| 1 mM EDTA | |||
| 1:500 | |||
| 20 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | |||
| height="15" | SDS | |||
| 10% w/v | |||
| 0.5% (w/v) | |||
| 1:20 | |||
| 500 uL | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | Proteinase K | |||
| 20 mg/mL | |||
| 500 ug/mL | |||
| 1:40 | |||
| 250 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | |||
| height="15" | nfH2O | |||
| - | |||
| - | |||
| - | |||
| Fill to 10 mL (~ mL) | |||
|} |
Revision as of 16:30, 1 April 2015
RNA FISH Based Cell Sorting
The goal of this protocol is to be able to use RNA FISH when doing cell sorting, which can be a potentially more valuable way of cell sorting than using antibody staining, as it provides greater discrimination prospects.
Protocol
NOTE: The original protocol (from the paper) is rather vague and several steps may require troubleshooting if my guesses as to their meaning are not correct.
RNA FISH
- Fix cells in 4% paraformaldehyde for 5 min
- Centrifuge cells at 1000g for 5 minutes
- Wash cells with 70% EtOH
- Ethanol permeabilization- 70% EtOH overnight at 4C
- Resuspend cells in RPHBwith labeled probes (0.5-1ng/uL)
- Incubate for 12 hrs at 30C
- Add equal volume of wash buffer and mix thoroughly
- Pellet by centrifugation (1000g for 5 min)
- Resuspend cells in wash buffer for 30 minutes at 30C
- Pellet by centrifugation and resuspend cells in wash buffer for 30 minutes at 30C
- Pellet cells and resuspend in flow buffer at 4C in preparation for sorting
Flow Cytometry, FACS, and RNA Extraction
- Sort by FACS into 4C sorting buffer
- Cell size was controlled by costaining with Hoeschst 33342 and selecting for diploid DNA content
- Pellet FACS sorted cells using 1000g for 5 minutes and resuspend in reverse cross-linking buffer at 50C for 1 hr
- Isolate total RNA via phenol-chloroform or other
Buffers
- RPHB
- 300 mM NaCl
- 30 mM sodium citrate
- 2.1M ammonium sulfate
- 10 mM EDTA
- 1 mg/mL E. coli tRNA
- 500 ug/mL BSA
- 25% v/v formamide
- pH 5.2
- Wash Buffer
- 25% v/v formamide
- 2X SSC buffer
- Flow Buffer
- 2X SSC
- Sorting Buffer
- 200mM NaCl
- 20 mM sodium citrate
- 1.5M ammonium sulfate
- 5 mM EDTA
- 2X SSC
- pH 5.2
- Reverse Cross-Linking Buffer
- 100mM NaCl
- 10mM Tris
- 1 mM EDTA
- 0.5% SDS (v/v)
- 500 ug/mL proteinase K
Buffer Prep
RPHB
Reagent | Stock Concentration | Final Concentration | Dilution | Volume in 10 mL |
SSC Buffer | 20X | 2X | 1:10 | 1 mL |
Ammonium Sulfate | NA, powder | 2.1 M | NA | 2.775g |
EDTA | 0.5 M | 10 mM | 1:50 | 200 uL |
E. coli tRNA | 20 mg/mL | 1 mg/mL | 1:20 | 500 uL |
BSA | 50 mg/mL | 500 ug/mL | 1:100 | 100 uL |
Formamide | 100% | 25% | 1:04 | 2.5 mL |
nfH2O | - | - | - | Fill to 10mL (~5 mL) |
Sorting Buffer
Reagent | Stock Concentration | Final Concentration | Dilution | Volume in 10 mL |
SSC Buffer | 20X | 2X | 1:10 | 1 mL |
Ammonium Sulfate | NA, powder | 1.5 M | NA | 1.982 g |
EDTA | 0.5 M | 5 mM | 1:100 | 100 uL |
nfH2O | - | - | - | Fill to 10mL (~9 mL) |
Reverse Cross-Linking Buffer
Reagent | Stock Concentration | Final Concentration | Dilution | Volume in 10 mL |
NaCl | NA, powder | 100 mM | - | 58.4 mg |
Tris | 1 M | 10 mM | 1:100 | 100 uL |
EDTA | 0.5 M | 1 mM EDTA | 1:500 | 20 uL |
SDS | 10% w/v | 0.5% (w/v) | 1:20 | 500 uL |
Proteinase K | 20 mg/mL | 500 ug/mL | 1:40 | 250 uL |
nfH2O | - | - | - | Fill to 10 mL (~ mL) |