TITLE: Ribosomal RNA Depletion and cDNA Synthesis
AUTHORS: Alicia Rich

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Ribosomal RNA depletion and cDNA synthesis using the Zymo-Seq RiboFree Universal cDNA Kit. This protocol should follow RNA isolation and purification using the Direct-zol™ RNA Microprep Kit (Zymo) and the RNA Clean & Concentrator Kit (Zymo).</div><div class = "text-block">We will follow the manufacturer’s instructions for reverse transcription thermocycling and ribosomal depletion.</div><div class = "text-block">After completing this protocol, proceed to qPCR using the PrimeTime Gene Expression Kit.</div><div class = "text-block">From the manufacturer: </div><div class = "text-block">This kit produces first-strand cDNA from any sample’s full transcriptome (both coding and non-coding). Overcome challenges in capturing mRNA from degraded and fragmented samples with this kit. In contrast to poly(A) targeted RNA pull-down or oligo (dT) priming, this kit produces cDNA from total RNA, including long-noncoding RNAs (lncRNA), intronic RNAs, nucleolar RNAs, and mRNAs with degraded poly(A) tails. </div><div class = "text-block">Ribosomal RNAs (rRNA) can comprise approximately 90% of the total cellular RNA and represents an obstacle when analyzing unique protein-coding sequences. Zymo Research’s RiboFreeTM Universal Depletion effectively removes rRNA and overrepresented transcripts (e.g. globin) without the use of probes that cause off-target digestion compared to popular rRNA removal kits. </div><div class = "text-block">RiboFreeTM Universal Depletion (rRNA, beta-globin) is compatible with all biological sample types, tissues (including whole blood), and does not require organism-specific probes. </div><div class = "text-block">A streamlined workflow minimizes user manipulation, resulting in a user-friendly protocol with little hands-on time. The RiboFreeTM Universal cDNA Kit is a simple and cost-effective method for generating first-strand cDNA with minimal bias compared to other rRNA depletion or poly(A) enrichment methods. </div></div>

[STEPS]
?. [0.0 Estimate Starting RNA concentration]
Estimate the starting RNA concentration of each extract using the Nanodrop.
?. [0.0 Estimate Starting RNA concentration]
Bring RNA extracts and RNase-free water on a cold block to the spectrophotometer downstairs.
on ice
?. [0.0 Estimate Starting RNA concentration]
Wash the sample reader with   and dry with a KimWipe.
?. [0.0 Estimate Starting RNA concentration]
Load  of elution water (blank) and initialize the system.
2 µl
?. [0.0 Estimate Starting RNA concentration]
Change the computer's setting to RNA and click the "blank" button.
?. [0.0 Estimate Starting RNA concentration]
Load   of sample and click the "measure" button.
2 µl
?. [0.0 Estimate Starting RNA concentration]
After the read is complete, record the A260/A280 and A260/A230 ratios as well as the amount of RNA recovered (in  ).
?. [0.0 Estimate Starting RNA concentration]
Wipe the sample reader with a clean, dry KimWipe between samples.
?. [0.0 Estimate Starting RNA concentration]
Interpret your results.
As a reference, collagen gels seeded with ~700,000 cells typically yield around 50-150 ng/µL of RNA when eluted in 30-35 µL of waterA lower than expected concentration of RNA indicates low cell numbers in the sample, poor homogenization of samples, or too much volume of water used in the elution step of RNA purification.Very pure RNA will have an A260/A280 ratio of ~2.1.  Anything higher than 1.8 is considered to be of acceptable purity, and a ratio of 260/A280 ratio is likely due to mixing phases when removing the upper aqueous phase of the Trizol separation or is also more common in samples with a very low yield of RNA.The A260/A230 ratio should also be above 2.0. A low A260/A230 ratio indicates contamination with the wash solutions, chaotropic salts, phenols, or protein.  A low A260/A230 ratio is most likely due to contamination of the samples with washing buffers during the spin column washes. Be more careful when handling the tubes, especially when adding a wash buffer or removing the spin-through. Try to gently pour out the flow-through and then carefully wipe away drops on the outer rim of the collection tube with a KimWipe.
?. [1.2 RiboFree Universal Depletion (Red Caps)]
Create the following thermocycler program for a total reaction volume of  .98 C3 minPre-Depletion Incubation68 C5 minPre-Depletion Incubation68 CHoldPre-Depletion Incubation68 CDepends on input - 30 min if >1 ug, 1 hr if >250 ng, or 2 hr if >100 ngDepletion Rxn68 CHoldDepletion Rxn98 C2 minStop Depletion25 CHoldStop Depletion> 1 ug30 min> 250 ng1 hr> 100 ng2 hrs3-16 hrs
50 µl
98 C3 minPre-Depletion Incubation68 C5 minPre-Depletion Incubation68 CHoldPre-Depletion Incubation68 CDepends on input - 30 min if >1 ug, 1 hr if >250 ng, or 2 hr if >100 ngDepletion Rxn68 CHoldDepletion Rxn98 C2 minStop Depletion25 CHoldStop Depletion
Refer to Appendix B of manufacturer protocol (table below) for further RiboFree Universal Depletion Incubation Times if troubleshooting is needed.
> 1 ug30 min> 250 ng1 hr> 100 ng2 hrs3-16 hrs
This universal depletion stage will involve transferring reagents to tubes while they are still inside the thermocycler. Make sure you are not overlapping with someone's scheduled use of the machine.
?. [1.1 First-Strand cDNA Synthesis (Yellow Caps)]
Create the following thermocycler program for a total reaction volume of :98 C3 minPrimer Annealing4 CHoldPrimer Annealing25 C5 minReverse Transcription48 C15 minReverse Transcription4 CHoldReverse Transcription
20 µl
98 C3 minPrimer Annealing4 CHoldPrimer Annealing25 C5 minReverse Transcription48 C15 minReverse Transcription4 CHoldReverse Transcription
?. [1.1 First-Strand cDNA Synthesis (Yellow Caps)]
Thaw the R1 and R2 Reagents. Mix thoroughly using one of the mini-vortexes or by flicking. Briefly spin down on the mini-centrifuge.
on ice
Avoid multiple freeze-thaws (make aliquots if necessary) and always keep samples on ice or cooling block during use.
?. [1.1 First-Strand cDNA Synthesis (Yellow Caps)]
Using RNase/RNase-Free Water, raise the volume of each RNA input sample to  in 0.2 mL PCR tubes or plates.
on ice
8 µl
?. [1.1 First-Strand cDNA Synthesis (Yellow Caps)]
Add   of the R1 Reagent to each sample for a total of  . Mix thoroughly using a mini-vortex. Briefly spin down using the mini-centrifuge.
on ice
2 µl
10 µl
For inputs  ,   of the R1 Reagent should be used. Supplement with DNase/RNase-free water.
?. [1.1 First-Strand cDNA Synthesis (Yellow Caps)]
Place tubes or plate in the thermocycler. Run Steps 1-2 (Primer Annealing Stage) of the program.98 C3 minPrimer Annealing4 CHoldPrimer Annealing
98 C3 minPrimer Annealing4 CHoldPrimer Annealing
?. [1.1 First-Strand cDNA Synthesis (Yellow Caps)]
Add  of the R2 Reagent to each sample.
[or just keep on 4C hold in thermocycler]
10 µl
?. [1.1 First-Strand cDNA Synthesis (Yellow Caps)]
Continue the thermocycler program through Steps 3-5 (Reverse Transcription) of the program.25 C5 minReverse Transcription48 C15 minReverse Transcription4 CHoldReverse Transcription
25 C5 minReverse Transcription48 C15 minReverse Transcription4 CHoldReverse Transcription
?. [1.2 RiboFree Universal Depletion (Red Caps)]
Thaw the D1, D2, and D3 Reagents. Before starting, mix thoroughly using the mini-vortex and then spin down briefly using the mini-centrifuge.
on ice
Avoid multiple freeze-thaws (make aliquots if necessary) and keep on ice after thawing between storage.
?. [1.2 RiboFree Universal Depletion (Red Caps)]
Add  of the D1 Reagent directly to each  sample for a total of . Mix thoroughly using the mini-vortex and then spin down briefly using the mini-centrifuge.
on ice
10 µl
20 µl
30 µl
?. [1.2 RiboFree Universal Depletion (Red Caps)]
Transfer the samples to the thermocycler and run Steps 1-3 (Pre-Depletion Incubation) of the "ribo depletion" program.
DO NOT REMOVE YOUR SAMPLES FROM THE THERMOCYCLER AT THE STEP 3 HOLD.
?. [1.2 RiboFree Universal Depletion (Red Caps)]
Without removing the tubes or plate, add   of the D2 Reagent to each  sample during the Step 3 hold for a total volume of  . Mix in the thermocycler by pipetting up and down.
10 µl
30 µl
40 µl
Optional: if available, you can use a multichannel pipette to minimize hands-on time for this step.
?. [1.2 RiboFree Universal Depletion (Red Caps)]
Close the thermocycler lid and continue through the Depletion Reaction of the "ribo depletion" program.
DO NOT REMOVE YOUR SAMPLES AT THE STEP 5 HOLD
?. [1.2 RiboFree Universal Depletion (Red Caps)]
Without removing the tubes, add   of the D3 Stop Reagent to each   sample during the Step 5 hold for a total volume of  . Mix in the thermocycler by pipetting.
10 µl
40 µl
50 µl
?. [1.2 RiboFree Universal Depletion (Red Caps)]
Close the thermocycler lid and continue through the Stop Depletion stage of the "ribo depletion" program. Remove samples from the thermocycler.
?. [1.2 RiboFree Universal Depletion (Red Caps)]
Add  of  ethanol to each  sample for a total volume of  . Mix by pipetting up and down.
on ice
25 µl
50 µl
75 µl
?. [Select-a-Size MagBead Clean-up]
Resuspend the magnetic particles immediately before use by vigorously inverting the Select-a-Size MagBeads until homogenous.
?. [Select-a-Size MagBead Clean-up]
Add  of Select-a-Size MagBeads to each sample. Mix thoroughly by pipetting until homogenous.
150 µl
?. [Select-a-Size MagBead Clean-up]
Incubate for
0 Room temperature
?. [Select-a-Size MagBead Clean-up]
Place the samples on a magnetic rack (included in kit) until the beads have fully separated from the solution. Then discard the supernatant.
Avoid aspirating any beads when removing the supernatant.
?. [Select-a-Size MagBead Clean-up]
While the beads are still on the magnetic rack, add  of  . Remove and discard the supernatant.
200 µl
?. [Select-a-Size MagBead Clean-up]
Aspirate any residual wash buffer when repeating the step (2 washes total).
?. [Select-a-Size MagBead Clean-up]
Remove the tubes from the magnetic rack and keep the tops open for  to dry the beads.
?. [Select-a-Size MagBead Clean-up]
Add ≥  of  to the beads and mix thoroughly by pipetting up and down until homogenous.
10 µl
?. [Select-a-Size MagBead Clean-up]
Incubate at  for  .
95 °C
?. [Select-a-Size MagBead Clean-up]
Place the tubes on a magnetic rack to separate the magnetic beads from the suspension, and transfer the eluate to a new tube.
Some beads may carry over into the eluate. Bead carry-over will not affect downstream reactions.