TITLE: RNA purification and cDNA synthesis 
AUTHORS: Vicki Deng, Fredrick leon, David Booth

[DESCRIPTION]
This protocol compiles multiple methods for purifying RNA from an S. rosetta lysate and provides a modified reverse transcriptase protocol to robustly synthesize cDNA from transcripts with higher GC content.

[STEPS]
SECTION: Culture cells for lysate
1. Grow enough culture for ~50x107 cells. 
for swimming cultures: seed 40 mLacclimated cells at ~8*10^4 cells/mlthen culture at 27 °C for 1440 min 
for thecate cultures: seed 100 mL acclimated cells in culture plate then culture at 27 °C for 1440 min 

Number of cells doesn't necessarily matter. You just want enough cells to extract sufficient nucleic acid from taking in account the loss of yield in subsequent steps.





SECTION: Culture cells for lysate
2. Follow preferential lysis protocol
SECTION: Extract RNA
3. You can extract total RNA (go to step 3.1) or mRNA (go to step 3.2)
SECTION: Extract RNA
3.1. Trizol LS total RNA extraction
Add 3:1 Trizol LS to lysate and incubate for 5 min
Add 200 µLchloroform to 750 µLof added Trizol LS and incubate for 3 min
Centrifuged sample at 12000 x g, 15 min, 4 °C
Collect aqueous layer

RNA precipitation
Add 500 µLcold isopropanol per 750 µL of added Trizol LS to collected aqueous layer and incubate on ice for 10 min
Centrifuge sample at12000 x g, 30 min, 4 °C
Washed pellet with cold 75% ethanol
Centrifuge sample at 7500 x g, 5 min, 4 °C
Resuspend with 1 millimolar (mM) ,pH 6.4.
Measure RNA concentration

or

RNeasy Cleanup
we use the RNeasy MinElute Cleanup Kit
1. Adjust the sample to a volume of100 µL
2. Add250 µL96–100% ethanol to the diluted RNA, and mix
3. Transfer the sample (700 µL) to an RNeasy MinElute spin column placed in a 2 ml collection tube. Centrifuge for 8000 x g, 15 s. Discard the flow-through.
4. Place the RNeasy MinElute spin column in a new 2 ml collection tube. Add 500 µLBuffer RPE to the spin column. Close the lid gently, and centrifuge for 8000 x g, 15 s 
5. Add500 µLof 80% ethanol to the RNeasy MinElute spin column. Close the lid gently, and centrifuge for 8000 x g, 2 min
6. Place the RNeasy MinElute spin column in a new 2 ml collection tube. Open the lid of the spin column, and centrifuge at full speed for5 min.
7. Place the RNeasy MinElute spin column in a new 1.5 ml collection tube. Add 14 µL RNase-free water directly to the center of the spin column membrane. Close the lid gently, and centrifuge for 1 min at full speed to elute the RNA
8. For long-term storage, supplement the RNA with 1 millimolar (mM) ,pH 6.4 












SECTION: Extract RNA
3.2. mRNA extraction
We use the NEB Magnetic mRNA Isolation Kit
Equilibrate 100 µLbeads with 200 µL binding buffer
Add lysate and mix for10 min
Pull down beads with magnet and remove supernatant
Add 500 µL Wash Buffer 1 and mix for 1 min
Pull down beads with magnet and remove supernatant
Repeat step 4-5
Add 500 µLWash Buffer 2 and mix for 1 min
Pull down beads with magnet and remove supernatant
Repeat step 7-8
Add 500 µL Low Salt Buffer and mix for 1 min
Pull down beads with magnet and remove supernatant
Add 20 µLelution buffer (can vary elution volume for desired concentration; can vary elution buffer, we have used Nano-pure water or nylon filtered 10 mM Tris-acetate pH 8.0)
Measure mRNA concentration
For long-term storage, supplement the RNA with 1 millimolar (mM) ,pH 6.4 











SECTION: cDNA synthesis
4. We use Invitrogen SuperScript™ IV Reverse Transcriptase kit

SECTION: cDNA synthesis
4.1. Anneal oligo d(T)20 primer to RNA sample
Assemble the reaction according to this table:


 

 2. Mix and incubate reaction at 65 °C for 5 min
 3. Place on ice for 1 min
SECTION: cDNA synthesis
4.2. Reverse transcription to make cDNA
Vortex 5x SSIV Buffer
To the annealed RNA templates from  , add the following components:

 
 4. Incubate reaction at 60 °C for 10 minutes (IMPORTANT: reaction temperature is increased from kit
     instructions to account for higher GC content in some transcripts)
 5. Inactivate reaction by incubation at 80 °Cor 10 min
 6. Remove RNA with incubation with 1 µL of RNase H at 37 °C for 20 min
SECTION: cDNA synthesis
5. Clone gene of interest with cDNA
Use generated cDNA in PCR reaction with gene specific primers. To verify that the RNA purfication and cDNA synthesis was successful, amplify a highly expressed transcript, such as cofillin (PTSG_01554).