> For the complete documentation index, see [llms.txt](https://salis-lab.gitbook.io/protocol-book/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://salis-lab.gitbook.io/protocol-book/test/rna/rna-extraction-for-promoter-libraries.md).
# Targeted RNA-Seq
## **RNA Extraction**
**Things to check before starting the protocol**
* **Make 1 mg/mL lysozyme in RNase-free 1x TE buffer \[prepare 150 µL per RNA sample]**
* **990 uL DEPC treated water**
* **10 uL 100x TE buffer**
* **(1.0 mg, Gram-negative) (3.0 mg, Gram-positive) lysozyme (from chicken egg)**
* **Store on ice**
* **Add 16 µL β-Mercaptoethanol (βME) to 1.6 mL Lysis Solution (Norgen Kit) in an RNase-free eppendorf tube and mix by quick vortexing \[prepare 400 µL per RNA sample required]**
* **Make two tubes.**
* **Additional Labeling and preparation**
* **Label 4 RNAse free eppendorf tubes for each biological sample.**
* **Label 1 purification column for each biological sample.**
* **Label 1 eppendorf tube for the loading dye master mix.**
**Steps to minimize RNAse contamination**
* **Cover hands, arms, head, and body with sterile protective gear (RNAses are in your saliva, your tears, and on your body surface).**
* **Wipe down all bench surfaces with RNAse Away**
* **Use only sterile, disposable plastic tubes, tips, and serological pipettes. It is important to keep a box set of tips for RNA use only -- do not use these tips for any other labwork, e.g. minipreps.**
**Protocol Steps**
1. **Spin down at room temperature 5 mL of bacterial culture at OD600 0.2 (log phase) at max rpm (4,750 rcf), and discard the supernatant \[try to withdraw as much supernatant as possible without disturbing the bacterial pellet]**
2. **Resuspend the pellet in 100 µL of the lysozyme solution (in TE) by pipetting up and down and/or vortexing and transfer to an RNase-free eppendorf tube. Incubate the resuspension at room temperature for (5 minutes, Gram-negative) (10 minutes, Gram-positive) \[if the room temperature is low, incubate at 25 °C]**
3. **Add 300 µL of the βME containing Lysis Solution (Buffer RL - Norgen Kit) and mix by vigorous vortexing for 10-15 seconds**
4. **Add 200 µL of ethanol (98-100%) to the lysate and mix by vigorous vortexing for 10-15 seconds \[use the EtOH bottle from the designated "RNA ONLY" drawer]**
5. **Assemble the RNA purification column and the collection tube (Norgen Kit) and add 600 µL of the lysate from Step 4 above to it. Spin at 14,000 rcf for 1 min at room temperature \[if all the lysate does not go through the column, spin for another 1 min]**
6. **Discard the flow-through and reassemble the column**
7. **Add 400 µL of the Wash Solution (Norgen Kit) to the column and spin at 14,000 rcf for 1 min at room temperature \[the Wash Solution should have been prepared beforehand by adding RNase-free ethanol to the Norgen supplied bottle]**
8. **Discard the flow-through and reassemble the column**
9. **Repeat Steps 7 & 8 two more times (total of 3 washes)**
10. **Spin the column at 14,000 rcf for 2 min at room temperature in order to dry the column resin**
11. **Discard the collection tube and place the column into a fresh RNase-free eppendorf tube \[use cold eppendorf tubes]**
12. **Add 50 µL of the Elution Solution (Norgen Kit) to the column and spin at room temperature at 200 rcf for 2 min, followed by 14,000 rcf for 1 min \[place the RNA on ice-water immediately and keep cold all the time]**
13. **For greater recovery, place the column back into the same eppendorf tube and repeat the elution with another 50 µL of the Elution Solution**
14. **To the rest of the 95 µL RNA, add 9.5 µL of the 10x TURBO DNase Buffer and 2 µL TURBO DNase (TURBO DNA-free™ Kit) and mix gently by flicking the tube**
15. **Incubate at 37 °C for 30 min**
16. **Add 10.7 µL DNase Inactivation Reagent (TURBO DNA-free™ Kit) and mix gently by flicking the tube \[before pipetting the DNase Inactivation Reagent, flick the tube a couple of times to ensure that the beads are uniformly dispersed]**
17. **Incubate at room temperature for 5-6 min while mixing gently by flicking the tube every 2 min**
18. **Spin at room temperature at 10,000 rcf for 1.5 min and transfer 100 µL of the supernatant to a fresh RNAse-free eppendorf tube \[use cold eppendorf tubes; keep cold all the time]**
19. **Take out a 3 µL aliquot in a separate RNase-free eppendorf tube for later analysis \[use cold eppendorf tubes; keep cold all the time]**
20. **Use the nanodrop to determine RNA concentration using the aliquots taken earlier in Steps 14 and 20 (pre- and post- DNAse treatment)**
21. **Run 1 µL each of the RNA samples from Steps 14 & 20 above (post- DNAse treatment) on a 1% agarose gel to check for RNA integrity.**
22. **Freeze down all the tubes at -80 °C.**
**Goal: 100 uL of total RNA at a concentration of 100 - 125 ng/uL. Total RNA extracted 10 -12.5 ug.**
## **RNA Clean-up and Concentration**
**This protocol uses the Qiagen RNeasy MinElute Cleanup Kit. It can purify up to 45 ug of RNA. Note: This kit does not retain RNA molecules below 200 nucleotides, which comprise 15-20% of Total RNA.**
**\*\*\*\*\*Use the two highest concentration tubes.\*\*\*\*\***
1. **Adjust sample volume to 100 uL. (Norgen Total RNA kit outputs 100uL)**
2. **Add 350 uL Buffer RLT and mix well.**
3. **Add 250 µl 96%–100% ethanol. Pipette to mix.**
4. **Transfer the sample mixture (\~700 µl) to an RNeasy MinElute spin column placed in a 2 ml collection tube.**
5. **Close the lid, and then centrifuge at 8000 × g for 15 seconds.**
6. **Discard the flow-through and collection tube.**
7. **Place the RNeasy MinElute spin column in a new 2 ml collection tube.**
8. **Add 500 µl Buffer RPE, and then close the lid.**
9. **Centrifuge at 8000 × g for 15 seconds.**
10. **Discard the flow-through and retain the collection tube.**
11. **Add 500 µl 80% ethanol to the RNeasy MinElute spin column, and then close the lid.**
12. **Centrifuge at 8000 × g for 2 minutes.**
13. **Without allowing the column to contact the flow-through, remove the RNeasy MinElute spin column from the collection tube.**
14. **Discard the flow-through and collection tube.**
15. **Place the RNeasy MinElute spin column in a new 2 ml collection tube.**
16. **Place the spin columns into the centrifuge with at least 1 empty position between columns, which avoids damaging the spin column lids. Orient the lids to point in a direction opposite the rotation of the rotor.**
17. **With the lids open, centrifuge at maximum speed for 5 minutes.**
18. **Discard the flow-through and collection tube.**
19. **Place the RNeasy MinElute spin column in a new 1.5 ml collection tube.**
20. **Add 14 uL of RNase-free water to the center of the spin-column membrane**
21. **Close the lid, and then centrifuge at maximum speed for 1 minute.**
1. **Expect to recover \~12 µl RNA.**
22. **Take 2 uL aliquot to determine the concentration and use with analytical gel.**
## **rRNA Depletion**
**This protocol will use the Illumina Ribo-Zero Removal Kit (MRZMB126). This kit can use a maximum of 5 ug Total RNA and a volume of 26 uL.**
**Prepare the Beads**
1. **Prepare the following consumables:**
| **Item** | **Storage** | **Instructions** |
| --------------------------------------- | -------------- | ------------------------------------------------------------ |
| **Magnetic beads** | **2°C to 8°C** | **Bring to room temperature. Vortex to mix** |
| **Magnetic Bead Resuspension Solution** | **2°C to 8°C** | **Bring to room temperature.** |
| **RNase-free water** | **2°C to 8°C** | **Thaw on ice. Invert to mix, and then centrifuge briefly.** |
**2. For each reaction, add 225 µl magnetic beads to a 1.5 ml microcentrifuge tube.**
**3. Place on a magnetic stand, with cap open, and wait until the liquid is clear (\~1 minute).**
**4. Remove and discard all supernatant.**
**5. Remove from the magnetic stand.**
**6. Wash 2 times as follows.**
1. **Add 225 µl RNase-free water.**
2. **Vortex to resuspend.**
**7. Place on a magnetic stand, with cap open, and wait until the liquid is clear (\~1 minute).**
**8. Remove and discard all supernatant.**
**9. Remove from the magnetic stand.**
**10. Add 65 µl Magnetic Bead Resuspension Solution.**
**11. Vortex to resuspend.**
**12. Set aside at room temperature.**
\
**Hybridize Probes**
1. **Prepare the following consumables:**
| **Item** | **Storage** | **Instructions** |
| ------------------------------ | ------------------ | ------------------------------------------------------------ |
| **Ribo-Zero Reaction Buffer** | **-85°C to -65°C** | **Thaw on ice. Invert to mix, and then centrifuge briefly.** |
| **Ribo-Zero Removal Solution** | **-85°C to -65°C** | **Thaw on ice. Invert to mix, and then centrifuge briefly.** |
| **RNase-free water** | **2°C to 8°C** | **Thaw on ice. Invert to mix, and then centrifuge briefly.** |
**2. Set a heat block or thermal cycler to 68°C.**
**3. For each sample, combine the following volumes in a 0.2 ml or 0.5 ml microcentrifuge tube. Pipette to mix.**
* **RNase-free water (26-x µl)**
* **Ribo-Zero Reaction Buffer (4 µl)**
* **RNA sample (x µl)**
* **Ribo-Zero Removal Solution (10 µl)**
**The total volume per sample is 40 µl.**
**4. Place on the preheated heat block or thermal cycler and incubate for 10 minutes.**
**5. Remove from heat, and then centrifuge briefly.**
**6. Incubate at room temperature for 5 minutes.**
**Remove rRNA**
1. **Set a heat block or thermal cycler to 50°C.**
2. **For each sample, do as follows.**
1. **Add 40 µl RNA sample to a 1.5 ml tube containing 65 µl washed magnetic beads. Immediately pipette to mix.**
2. **Vortex for 10 seconds, and then set aside at room temperature.**
**3. Incubate at room temperature for 5 minutes.**
**4. Place on the preheated heat block or thermal cycler and incubate for 5 minutes.**
**5. Immediately place on a magnetic stand, with cap open, and wait until the liquid is clear (\~1 minute).**
**6. Transfer 85–90 µl supernatant containing depleted RNA to a fresh 1.5 ml tube.**
**7. Set aside on ice.**
\
**Clean Up Depleted RNA**
-------------------------
**This protocol will use Qiagen RNeasy MinElute Cleanup Kit (74204).**
1. **Prepare fresh 80% ethanol from absolute ethanol. Set aside both forms of ethanol on ice.**
2. **Adjust the sample to a volume of 100 μl with RNase-free water (Add 10-15 uL water).**
3. **Add 350 uL Buffer RLT and mix well.**
4. **Add 550 µl 96%–100% ethanol. Pipette to mix.**
5. **Add half the sample mixture (\~500 µl) to an RNeasy MinElute spin column placed in a 2 ml collection tube.**
6. **Close the lid, and then centrifuge at 8000 × g for 15 seconds.**
7. **Discard the flow-through.**
8. **Add the remaining sample to the spin column.**
9. **Repeat steps 6–7.**
10. **Discard the flow-through and collection tube.**
11. **Place the RNeasy MinElute spin column in a new 2 ml collection tube.**
12. **Add 500 µl Buffer RPE, and then close the lid.**
13. **Centrifuge at 8000 × g for 15 seconds.**
14. **Discard the flow-through and retain the collection tube.**
15. **Add 500 µl 80% ethanol to the RNeasy MinElute spin column, and then close the lid.**
16. **Centrifuge at 8000 × g for 2 minutes.**
17. **Without allowing the column to contact the flow-through, remove the RNeasy MinElute spin column from the collection tube.**
18. **Discard the flow-through and collection tube.**
19. **Place the RNeasy MinElute spin column in a new 2 ml collection tube.**
20. **Place the spin columns into the centrifuge with at least 1 empty position between columns, which avoids damaging the spin column lids. Orient the lids to point in a direction opposite the rotation of the rotor.**
21. **With the lids open, centrifuge at maximum speed for 5 minutes.**
22. **Discard the flow-through and collection tube.**
23. **Place the RNeasy MinElute spin column in a new 1.5 ml collection tube.**
24. **Add 14 uL of RNase-free water to the center of the spin-column membrane**
25. **Close the lid, and then centrifuge at maximum speed for 1 minute.**
1. **Expect to recover \~12 µl RNA.**
26. **Take 2 uL aliquot to measure RNA concentration and for gel.**
## **Reverse Transcriptase Reaction and RNase H Degradation**
**This protocol will use Invitrogen’s SuperScript IV First-Strand Synthesis System**
1. **Add the following to a PCR reaction tube.**
| **Component** | **Volume** |
| -------------------------------------------------- | ---------- |
| **2 μM gene-specific reverse primer** | **1 uL** |
| **10 mM dNTP mix (10 mM each)** | **1 uL** |
| **Template RNA (10 pg–500 ng mRNA) (up to 11 uL)** | **10 uL** |
| **DEPC-treated water (up to 13 uL)** | **1 uL** |
1. **Pipet mix and briefly centrifuge the components.**
2. **Heat the RNA-primer mix at 65 C for 5 min, then put on ice for a least 1 min.**
3. **Make the following master mix.**
| **Component** | **1 RXN amounts** | **3 RXN amounts** |
| ---------------------------------------------------- | ----------------- | ----------------- |
| **5× SSIV Buffer** | **4 uL** | **12 uL** |
| **100 mM DTT** | **1 uL** | **3 uL** |
| **Ribonuclease Inhibitor** | **1 uL** | **3 uL** |
| **SuperScript™ IV Reverse Transcriptase (200 U/μL)** | **1 uL** | **3 uL** |
**5. Pipet mix and briefly centrifuge the components.**
**6. Add 7 uL of RT reaction master mix to each annealed RNA.**
**7. Incubate the mix at 50 C for 10 min.**
**8. Inactivate the reaction by incubating at 80 C for 10 min.**
**9. Add 1 uL of RNAse H and incubate at 37 C for 20 min.**
**10. Congrats, you have ss,cDNA!**
## **PCR Amplification**
