Optimized protocol for translatome analysis of mouse brain endothelial cells
Won-Jong OH, Namsuk Kim, Mi-Hee Jun, Jin-Young Jeong
Abstract
Brain endothelial cells (BECs) are important conduits that deliver oxygen and nutrients, protect parenchyma cells from toxins, and drain wastes to maintain brain homeostasis. Impairment of BECs has been implicated in diverse neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Therefore, molecular analysis of BECs is important for understanding the molecular pathogenesis of these neurological diseases. Even though many transcriptome analyses for BECs have been developed, mRNA levels do not necessarily correlate with the levels of actively translated proteins. Translatome analysis using RiboTag mice, in which Rpl22 , a ribosomal component, is tagged by the hemagglutinin epitope under Cre recombinase activation, could serve as an excellent tool that overcomes these caveats.However, implementation of this technique is limited by high noise-to-signal ratios as well as the low yield of mRNAs from BECs, which limits bulk gene expression analysis. In this study, we established a protocol to isolate highly pure mRNAs from BECs in the cortex of eight- to twelve-week-old male Tie2-Cre; Rpl22HA/HA HA/HA mice by using a cell strainer to trap blood vessels prior to immunoprecipitation. According to the results of RT–PCR, the specificity of the mRNA pools isolated by our protocol was much higher than that of the pools isolated by the standard protocol. We were also able to generate a high-quality cDNA library for RNA-seq with the small amount of mRNA isolated with our protocol. Thus, this optimized method will be useful for future studies of BECs at the molecular level.
Before start
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An RNase -free environment is essential. Use barrier pipet tips to avoid RNase contamination. Wipe down the surface of an experimental table and all equipment including surgical tools, pipets, etc., with RNase Zap.
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Homogenization buffer and high-salt buffer should be freshly prepared.
Steps
Vessel isolation
The whole mouse cortex of a Tie2-Cre; Rpl22HA/HA HA/HA mouse is isolated in the chilled DMEM. Then, tissues are dissociated by using a glass homogenizer (WHEATON, 357542) in 10mL of chilled DMEM.
1000x g,4°C
After discarding the supernatants, the pellets are resuspended in 15mL of 20 % BSA-DMEM to avoid myelin contamination.
2500x g,4°C
After discarding the supernatants, the pellets are resuspended in 5mL of chilled PBS.
PBS containing blood vessels is passed through a 40-micrometer cell strainer.
Immunoprecipitation
The strainer mesh containing vessels is then cut with a disposable scalpel (Bard-Parker, 371611) and transferred into a microcentrifuge tube for lysis in 600µL of homogenization buffer containing 1% (v/v) NP-40, 100millimolar (mM) KCl, 50millimolar (mM) Tris (7.4), 12millimolar (mM) MgCl2, cycloheximide (100mg/mL ), heparin (1mg/mL), Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, 78444), RNA inhibitor (5 units/ml, Promega, N2615), and 1millimolar (mM) DTT).
The lysates are incubated On ice for 0h 5m 0s .
12000x g,4°C
After being transferred to a new 1.5 ml microcentrifuge tube, the supernatants are incubated with a mouse monoclonal antibody against the HA epitope tag (1:200, Millipore, 05-904) for 4h 0m 0s at 4°C with rotation by using a multimixer (NanoEnTek, 4519).
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Protein A/G magnetic beads (Thermo Fisher Scientific, 88803) equilibrated in homogenization buffer for 30 min are added to the antibody-lysate solution and incubated at 4°C with gentle rotation.
The next day, after a brief spin-dwon, the magnetic beads are washed five times with 1mL high salt buffer ( 1% NP-40, 300millimolar (mM) KCL, 50millimolar (mM) Tris (7.4 ), 12millimolar (mM) MgCl2, cycloheximide (100mg/mL ), and 0.5millimolar (mM) DTT).
mRNA isolation
After the last wash, all supernatants are removed and 1mL of TRIzol reagent (Invitrogen, 15596026) is added to the bead-antibody-tissue homogenate, followed by 200µL of chloroform (Sigma-Aldrich, C2432).
12000x g,4°C
The upper aqueous layer (approximately 600µL) is transferred into a new 15 ml conical tube, and 60µL of 4Molarity (M) LiCl, 120µL of 20 X TE (0.2Molarity (M) Tris-HCl, 20millimolar (mM)EDTA, 7.5, Promega, A2651), 1.8mL of 100% ethyl alcohol (Sigma, E7023), and 3mL of glycogen (Roche, 10901393001) are added for RNA precipitation.
The mRNA mixture is incubated at -20°C
The following day, samples are centrifuged 12000x g,4°C. After the supernatants are discarded, 1mL of 75% ethyl alcohol is added to the pellets for washing.
After centrifugation7500x g,4°C and subsequent supernatant removal, the samples are air-dried for 0h 5m 0s at Room temperature. Do not overdry the beads.
The dried pellets are then resuspended in 16µL of RNase-free water.
2µL of DNase I and 2µL of 10X DNase I Reaction Buffer (Invitrogen, 18068-015) are added to the reaction mixture, which is then incubated for 0h 15m 0s at Room temperature .
DNaseI is inactivated by adding 25millimolar (mM) of EDTA and heating at 65°C for 0h 10m 0s .
For RNA precipitation, 2.2µL of 4Molarity (M) of LiCl, 4.8µL of 20 X TE (0.2Molarity (M) Tris-HCl, 20millimolar (mM) EDTA, 7.5(Promega, A2651), 66µL of 100% ethyl alcohol (Sigma-Aldrich, E7023), and 1µL of glycogen (Roche, 10901393001) are added to the RNA mixture, followed by incubation at -20°C .
The next day, the RNA mixture is centrifuged 12000x g,4°C
After removing the supernatants, 1mL of 75% ethyl alcohol is added to the pellets for washing.
After centrifugation 7500x g,4°C , the supernatants are discarded.
The pellets are then air-dried and finally resuspended in 10µL of RNase-free water.
Generation of cDNA library
The amount of isolated mRNA is measured by using High Sensitivity RNA ScreenTape (Agilent, 5067-5579), High Sensitivity RNA ScreenTape Reagent(Agilent, 5067-5580), and a High Sensitivity RNA ScreenTape ladder(Agilent, 5067-5581) from the Agilent 4200 TapeStation System according to the manufacturer's instructions.
One nanogram of mRNA obtained from RiboTag immunoprecipitation is reverse-transcribed into cDNA using the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (NEB, E6420L) according to the manufacturer’s protocol.
One nanogram of mRNA is added to the mixture containing 1µL of NEBNext Single Cell RT (Reverse Transcription) Primer Mix. The 9µL of the final volume is achieved by adding nuclease-free water.
The mixture is incubated at 70°C for 0h 5m 0s with the heated lid set to 105°C for annealing and then held at 4°C.
The RT mixture is prepared in a separate tube as follows On ice ; 5µL of NEBNext Single Cell RT buffer, 1µL of NEBNext Template Switching Oligo, 2µL of NEBNext Single Cell RT Enzyme Mix, 3µL of nuclease-free water. It is important to vortex the NEBNext Single Cell RT buffer prior to use for optimal performance.
The RT mixture (11µL) is combined with the annealed sample (9µL ). Mix well by pipetting up and down at least 10 times.
The reaction is incubated in a thermocycler with the following steps: the heated lid is set to 105°C, followed by 1h 30m 0s at 42°C and 0h 10m 0s at 70°C , and then held at 4°C
The cDNA amplification mix is prepared as follows: 50µL of NEBNext Single Cell cDNA PCR Master Mix, 2µL of NEBNext Single Cell cDNA PCR Primer, and 28µL of nuclease-free water.
80µL of cDNA amplification mix are added to 20µL of the sample with pipetting.
The reaction is performed in a thermocycler with the following PCR cycling conditions.
| A | B | C | D |
|---|---|---|---|
| Initial Denaturation | 98 °C | 45 sec | 1 |
| Denaturation | 98 °C | 10 sec | 32 |
| Annealing | 62 °C | 15 sec | |
| Extension | 72 °C | 3 min | |
| Final Extension | 72 °C | 5 min | 1 |
| Hold | 4 °C |
For the next step, the NEBNext Bead Reconstitution Buffer and the SPRI (Solid Phase Reversible Immobilization) beads should be warmed to Room temperature for at least 0h 30m 0s before use.
60µL SPRI beads are added to the PCR. (mix well by pipetting up and down at least 10 times).
The samples are incubated for at least 0h 5m 0s at Room temperature.
The samples are placed on the magnetic stand (Promega, Z5342) to separate the beads from the supernatant.
After 0h 5m 0s, the supernatant is removed. then, 200µL of 80% freshly prepared ethanol is added for washing. The samples are Incubated at Room temperature for 0h 0m 30s, and then the supernatant is carefully removed and discarded.
. This process is repeated twice. The samples are air-dried for 0h 5m 0s at Room temperature. Do not overdry the beads.
50µL of 0.1X TE (diluted from 1X TE buffer) is added to the samples to elute the cDNA from the beads.
The samples are mixed well and incubateed for at least 0h 2m 0s at Room temperature.
Next, 45µL of NEBNext Bead Reconstitution Buffer is added to the cDNA-Bead mixture. Mix well by pipetting up and down at least 10 times and incubate for at least 0h 5m 0s at Room temperature.
The samples are placed on a magnetic stand to separate the beads.
After 0h 5m 0s , the supernatant is carefully removed.
Then, 200µL of 80% freshly prepared ethanol is added to the tube to wash the beads. After 0h 0m 30s of incubation at Room temperature , . This process is repeated twice.
The beads containing cDNA are air-dried for 0h 5m 0s at Room temperature. Do not overdry the beads.
cDNA is eluted from the beads by adding 33µL of 1X TE. Mix well by pipetting up and down at least 10 times. The sample is incubated for at least 0h 2m 0s at Room temperature.
The sample is placed on the magnetic stand. After 0h 5m 0s of incubation at Room temperature, 30µL of the solution is transferred to a new tube.
The cDNA quality and quantity can be assessed by using High Sensitivity D5000 ScreenTape (Agilent, 5067-5592), High Sensitivity D5000 ScreenTape Reagent (Agilent, 5067-5593), and a High Sensitivity D5000 ScreenTape ladder (Agilent, 5067-5594) in the Agilent 4200 TapeStation System.
40ng of cDNA is used for Illumina NGS (Next Generation Sequencing) library preparation.
40ng of cDNA in 1X TE is mixed with 7µL of NEBNext Ultra II FS Reaction Buffer and 2µL of NEBNext Ultra II FS Enzyme Mix in a PCR tube. The final volume of the mixture is brought to 35µL, and the sample is vortexed for 0h 0m 5s.
In a thermocycler, with the heated lid set to 75°C, the following program is performed: 0h 25m 0s at 37°C and 0h 30m 0s at 65°C.
While the PCR is running, prepare the solution for the next step. NEBNext Adaptor for Illumina is diluted by 25-fold in the NEBNext Adaptor Dilution Buffer.
The following components should be added directly to the above sample (35µL). The adaptor should be added separately to each sample (DO NOT premix with ligation master mix and enhancer).
| A | B |
|---|---|
| FS Reaction Mixture | 35 μl |
| NEBNExt Ultra II Ligation Master Mix | 30 μl |
| NEBNext Ligation Enhancer | 1 μl |
| NEBNext Adaptor for Illumina (dilluted 1:25) | 2.5 μl |
The samples are mixed well by using pipetting the entire volume up and down at least 10 times. The ligation mixture is incubated at 20°C for 0h 15m 0s in a thermocycler without the heated lid.
3µL of USER Enzyme, a mixture of uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase endonuclease VIII, is added to the ligation mixture and mixed well. Incubate at 37°C for 0h 15m 0s .
For the next step, the NEBNext Bead Reconstitution Buffer and the SPRI beads should be warm to Room temperature for at least 0h 30m 0s before use.
57µL of SPRI beads are added to the PCR reaction. The sample is incubated for at least 0h 5m 0s at Room temperature.
The sample is placed on a magnetic stand.
After 0h 5m 0s incubation, the supernatant is removed. Then, 200µL of 80% freshly prepared ethanol is added to the tube. After incubation at Room temperature for 0h 0m 30s, the supernatant is removed. . This process is repeated twice.
The beads containing cDNA are air-dried for 0h 5m 0s at Room temperature . Do not overdry the beads.
17µL of 0.1X TE is added to resuspend the beads. The cDNA-bead mixture is incubated for at least 0h 2m 0s at Room temperature.
The sample is placed on a magnetic stand. After 0h 5m 0s, 15µL of the cleared solution is transferred to a new PCR tube.
The following components are combined into a new PCR tube.
| A | B |
|---|---|
| Adaptor Ligated DNA Fragments | 15 μl |
| NEBNext Ultra II Q5 Master Mix | 25 μl |
| Index Primer / i7 | 5 μl |
| Index Primer / i5 | 5 μl |
Labelling with dual barcodes is performed by using the following PCR cycling conditions.
| A | B | C | D |
|---|---|---|---|
| Initial Denaturation | 98 °C | 30 sec | 1 |
| Denaturation | 98 °C | 10 sec | 8 |
| Annealing | 65°C | 75 sec | |
| Final Extension | 65 °C | 5 min | 1 |
| Hold | 4 °C |
For the next step, the NEBNext Bead Reconstitution Buffer and the SPRI beads should be warmed to Room temperature for at least 0h 30m 0s before use.
The PCR mixture is resuspended in 45µL of SPRI beads. The sample is incubated for at least 0h 5m 0sat Room temperature.
The cDNA-bead mixture is placed on a magnetic stand to separate the beads from the supernatant.
After 0h 5m 0s, the supernatant is removed and discarded.
200µL of 80% freshly prepared ethanol are added to the tube in the magnetic stand. . This process is repeated twice.
The beads containing cDNA are air-dried on a magnetic stand for 0h 5m 0s at Room temperature.
The cDNA library is eluted by adding 33µL of 0.1X TE. Mix well by pipetting up and down 10 times.
The sample is placed on a magnetic stand. After 0h 5m 0s, 30µL of the sample containing the cDNA library is transferred to a new tube. Libraries can be stored at -20°C.
Before NGS, the quality of the final cDNA libraries is checked by using High Sensitivity D1000 ScreenTape (Agilent, 5067-5584), High Sensitivity D1000 ScreenTape Reagent(Agilent, 5067-5585), and a High Sensitivity D1000 ScreenTape ladder (Agilent, 5067-5587) in the Agilent 4200 TapeStation System.
