RNA Extraction
Reagents Needed:
Trizol (ThermoFisher, 15596018)
Chloroform
Glycoblue or other glycogen (ThermoFisher, AM9516)
Isopropanol
Ethanol
SUPERaseIN (ThermoFisher, AM2696)
Nuclease-free water (ThermoFisher, AM9939)
1)Samples typically start in 0.5-1.0 mL of Trizol. To extract total RNA, first thaw the sample at room temperature. Once the sample reaches room temperature, allow it to stand at room temperature for 5 minutes.
2)Add 200 uL of chloroform for every 1 mL of Trizol to the sample and shake by hand for 15 s. Allow the sample to stand at room temperature for 3 minutes.
3)Centrifuge the sample at 12,000 x g for 15 minutes in the cold room.
4)The aqueous phase will form on the top of the sample after centrifugation. Remove the aqueous phase (typically 300-500 uL) carefully with a P200 and dispense in an RNase-free non-stick tube. Avoid pipetting any white material at the interface between the aqueous and organic phases- this contains DNA.
5)Add 3 uL of GlycoBlue (15 ug/uL stock) to the aqueous sample. Add 0.5 mL of isopropanol to the aqueous sample for every 1 mL of Trizol in the original sample from step 1). Vortex the sample to mix the isopropanol into the aqueous sample.
6)Incubate the sample at -80C for at least 1 hour.
7)Place the sample on ice and centrifuge at 12,000 x g for 10 minutes in the cold room. A visible, blue-white pellet should form.
8)Remove the supernatant by aspiration and wash the pellet with 1 mL of 75% ethanol for every 1 mL of Trizol in the original sample. It’s OK if the pellet detaches from the tube.
9)Centrifuge the sample at 12,000 x g for 5 minutes in the cold room.
10)Remove the supernatant by aspiration and allow the sample to stand at room temperature to dry the pellet.
11)Re-suspend the dry pellet in 20 uL of Nuclease-free water with 0.5 uL of SUPERaseIN.
mRNA Isolation (Start here if using previously isolated total RNA)
Reagents Needed:
Wash Buffer: 20 mM Tris pH 8.0, 50 mM NaCl, 0.1% Tween-20
Hybridization Buffer: 20 mM Tris pH 8.0, 1 M NaCl, 0.1% Tween-20
Dynabeads™ MyOne™ Streptavidin C1 (ThermoFisher, 65001)
Biotinylated LNA-Oligo(dT) (Exiqon, 300100-03)
SUPERaseIN (ThermoFisher, AM2696)
Nuclease-free water (ThermoFisher, AM9939)
1)For each total RNA sample, take 20 uL of streptavidin-coated C1 Dynabeads (10 mg/mL stock). Wash the beads 2x in 0.5 mL of Wash Buffer. Wash the beads 1x in 20 uL of Hybridization Buffer. Resuspend in 20ul of Hybridization Buffer.
2)For every 20 uL of beads, add 4 uL of 25 uM biotinylated LNA-Oligo(dT). Incubate at room temperature for 15 minutes on a rotisserie.
3)Wash the beads 2x in 0.5 mL of Wash Buffer. Wash the beads 1x in 20 uL of Hybridization Buffer. Resuspend beads in 20ul Hybridization Buffer.
4)Add 20 uL of LNA-Oligo(dT)-coated beads in Hybridization Buffer to each sample (re-suspended pellet from step 11) of the previous section).
5)Incubate the sample-bead mixture at room temperature on a rotisserie for 45 minutes. Turn heat block to 75°C for step 7.
6)Wash the beads 3x with Wash Buffer. Re-suspend the beads in 15 uL of RNAse-free water with 0.5 uL of SUPERaseIN.
7)Incubate the beads at 75°C for 2 minutes. Immediately place tube on magnet and let the beads separate from supernatant.Immediately remove the supernatant containing eluted mRNA. Place the eluted mRNA on ice in an RNase-free, non-stick tube. NOTE: This step should be done one sample at a time and very carefully in order to not lose the mRNA.
Library Preparation
Reagents Needed:
SMARTer® Stranded RNA-Seq Kit (Clontech, 634836 12 rxn)
AMPureXP beads (Beckman Coulter, A63880)
Follow the instructions in the Clontech SMARTer Stranded RNA-Seq kit.
Clontech protocol:
Fragmentation: 4 min
After reverse transcription and before PCR, follow the instructions for double purification of the cDNA through two rounds of AMPure XP beads.
PCR Notes: The PCR is performed with the beads from the previous cleanup step still in the reaction. In order to optimize the PCR step, after the second cleanup resuspend the beads in 44ul nuclease-free water. Move 22ul to new PCR tube, keep remaining 22ul on ice or at 4°C. Make the rest of the PCR master mix separately (EXCLUDING water and Reverse PCR primer). Add 1ul unique Reverse PCR primer to each reaction. Add 27ul of PCR master mix (SeqAmp buffer, universal forward primer, SeqAmp DNA polymerase) and mix well. Perform PCR as described based on input RNA. After PCR cleanup and Bioanalyzer/Qubit, if it’s too low, redo the PCR with more cycles. If it has a double peak, too many cycles of PCR were performed. If necessary, use the remaining 22ul sample to redo the PCR with the correct number of cycles.
Sequencing
Reagents Needed:
NaOH
Tris-HCl pH 7
PhiX (Illumina, FC-110-3001)
NextSeq High Output 75 cycles (Illumina, FC-404-2005)
Use the High Output 75 cycle NextSeq kit for sequencing (1x75bp plus indexing). Samples can be loaded at 1.8pM with 5% PhiX.