TITLE: Optimized protocol for translatome analysis of mouse brain endothelial cells
AUTHORS: Won-Jong OH, Namsuk  Kim, Mi-Hee Jun, Jin-Young Jeong

[DESCRIPTION]
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 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]
1. 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.

2. Homogenization buffer and high-salt buffer should be freshly prepared.


[GUIDELINES]
1. 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.


2. Homogenization buffer and high-salt buffer should be freshly prepared.

3. Washes should be done in cold conditions. 

4. Tissue samples should be processed fresh directly to RNA yield.

5. The average amount of BEC mRNA => whole cortex ( 8-12 weeks): 7.3 ng, visual cortex ( 8-12 weeks): 1.05 ng, visual cortex (2 weeks): 0.25 ng per mouse.


[STEPS]
SECTION: Vessel isolation
1. The whole mouse cortex of a Tie2-Cre; Rpl22HA/HA mouse is isolated in the chilled DMEM. Then, tissues are dissociated by using a glass homogenizer (WHEATON, 357542) in 10 mL of chilled DMEM. 



SECTION: Vessel isolation
2. 1000 x g, 10 min, 4 °C 

SECTION: Vessel isolation
3. After discarding the supernatants, the pellets are resuspended in 15 mL of 20 % BSA-DMEM to avoid myelin contamination. 


SECTION: Vessel isolation
4. 2500 x g, 15 min, 4 °C 

SECTION: Vessel isolation
5.   

SECTION: Vessel isolation
6. After discarding the supernatants, the pellets are resuspended in 5 mL  of chilled PBS. 

SECTION: Vessel isolation
7. PBS containing blood vessels is passed through a 40-micrometer cell strainer. 

SECTION: Immunoprecipitation
8. The strainer mesh containing vessels is then cut with a disposable scalpel (Bard-Parker, 371611) and transferred into a microcentrifuge tube for lysis in 600 µL of homogenization buffer containing 1 % (v/v)  NP-40,  100 millimolar (mM) KCl, 50 millimolar (mM)  Tris (pH 7.4), 12 millimolar (mM)  MgCl2, cycloheximide (100 mg/mL ), heparin (1 mg/mL), Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, 78444), RNA inhibitor (5 units/ml, Promega, N2615), and 1 millimolar (mM)  DTT).

SECTION: Immunoprecipitation
9. The lysates are incubated on ice  for 5 min .

SECTION: Immunoprecipitation
10. 12000 x g, 10 min, 4 °C 

SECTION: Immunoprecipitation
11. 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 240 min  at 4 °C  with rotation by using a multimixer (NanoEnTek, 4519).
.

SECTION: Immunoprecipitation
12. 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.
SECTION: Immunoprecipitation
13. The next day, after a brief spin-dwon, the magnetic beads are washed five times with 1 mL  high salt buffer ( 1% NP-40, 300 millimolar (mM) KCL, 50 millimolar (mM)  Tris (pH 7.4 ), 12 millimolar (mM) MgCl2, cycloheximide (100 mg/mL ), and 0.5 millimolar (mM)  DTT). 

SECTION: mRNA isolation
14. After the last wash, all supernatants are removed and 1 mL of TRIzol reagent (Invitrogen, 15596026) is added to the bead-antibody-tissue homogenate, followed by 200 µL of chloroform (Sigma-Aldrich, C2432). 
 




SECTION: mRNA isolation
15. 12000 x g, 10 min, 4 °C 

SECTION: mRNA isolation
16. The upper aqueous layer (approximately  600 µL) is transferred into a new 15 ml conical tube, and 60 µL  of 4 Molarity (M)  LiCl,  120 µL  of 20 X TE (0.2 Molarity (M) Tris-HCl, 20 millimolar (mM)EDTA, pH 7.5, Promega, A2651), 1.8 mL  of 100% ethyl alcohol (Sigma, E7023), and 3 mL of glycogen (Roche, 10901393001) are added for RNA precipitation. 

SECTION: mRNA isolation
17. The mRNA mixture is incubated   at -20 °C 

SECTION: mRNA isolation
18. The following day, samples are centrifuged 12000 x g, 10 min, 4 °C. After the supernatants are discarded, 1 mL  of  75%  ethyl alcohol is added to the pellets for washing. 

SECTION: mRNA isolation
19. After centrifugation7500 x g, 5 min, 4 °C and subsequent supernatant removal, the samples are air-dried for 5 min at Room temperature. Do not overdry the beads.
SECTION: mRNA isolation
20. The dried pellets are then resuspended in 16 µL  of RNase-free water. 

SECTION: mRNA isolation
21. 2 µL of  DNase I and 2 µL of 10X DNase I Reaction Buffer (Invitrogen, 18068-015) are added to the reaction mixture, which is then incubated for 15 min at Room temperature .

SECTION: mRNA isolation
22. DNaseI is inactivated by adding 25 millimolar (mM) of EDTA and heating at 65 °C for  10 min .

SECTION: mRNA isolation
23. For RNA precipitation, 2.2 µL  of 4 Molarity (M) of LiCl, 4.8 µL  of 20 X TE (0.2 Molarity (M)  Tris-HCl, 20 millimolar (mM)  EDTA, pH 7.5(Promega, A2651), 66 µL  of 100%  ethyl alcohol (Sigma-Aldrich, E7023), and 1 µL of glycogen (Roche, 10901393001) are added to the RNA mixture, followed by   incubation at  -20 °C . 

SECTION: mRNA isolation
24. The next day, the RNA mixture is centrifuged 12000 x g, 10 min, 4 °C 

SECTION: mRNA isolation
25. After removing the supernatants, 1 mL  of 75% ethyl alcohol is added to the pellets for washing. 

SECTION: mRNA isolation
26. After centrifugation  7500 x g, 5 min, 4 °C , the supernatants are discarded. 

SECTION: mRNA isolation
27. The pellets are then air-dried and finally resuspended in 10 µL  of RNase-free water.

SECTION: Generation of cDNA library
28. 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.
SECTION: Generation of cDNA library
29. 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.
SECTION: Generation of cDNA library
30. One nanogram of mRNA is added to the mixture containing 1 µL of NEBNext Single Cell RT (Reverse Transcription) Primer Mix. The 9 µL  of the final volume is achieved by adding nuclease-free water. 

SECTION: Generation of cDNA library
31. The mixture is incubated at 70 °C  for 5 min  with the heated lid set to 105 °C  for annealing and then held at 4 °C.

SECTION: Generation of cDNA library
32. The RT mixture is prepared  in a separate tube as follows on ice ; 5 µL of NEBNext Single Cell RT buffer, 1 µL of NEBNext Template Switching Oligo, 2 µL of NEBNext Single Cell RT Enzyme Mix, 3 µL of nuclease-free water. It is important to vortex the NEBNext Single Cell RT buffer prior to use for optimal performance. 

SECTION: Generation of cDNA library
33. The RT mixture (11 µL) is combined with the annealed sample (9 µL ). Mix well by pipetting up and down at least 10 times. 

SECTION: Generation of cDNA library
34. The reaction is incubated in a thermocycler with the following steps: the heated lid is set to 105 °C, followed by 90 min  at 42 °C and 10 min  at 70 °C , and then held at 4 °C 

SECTION: Generation of cDNA library
35. The cDNA amplification mix is prepared as follows: 50 µL of NEBNext Single Cell cDNA PCR Master Mix, 2 µL of NEBNext Single Cell cDNA PCR Primer, and 28 µL of nuclease-free water.

SECTION: Generation of cDNA library
36. 80 µL of cDNA amplification mix are added to 20 µL  of the sample with pipetting.

SECTION: Generation of cDNA library
37. The reaction is performed in a thermocycler with the following PCR cycling conditions.

 


SECTION: Generation of cDNA library
38. 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 30 min  before use.

SECTION: Generation of cDNA library
39. 60 µL  SPRI beads are added to the PCR. (mix well by pipetting up and down at least 10 times).

SECTION: Generation of cDNA library
40. The samples are incubated for at least 5 min  at Room temperature.

SECTION: Generation of cDNA library
41. The samples are placed on the magnetic stand (Promega, Z5342) to separate the beads from the supernatant. 
SECTION: Generation of cDNA library
42. After 5 min, the supernatant is removed. then, 200 µL  of 80% freshly prepared ethanol is added for washing. The samples are Incubated at Room temperature  for 30 s,  and then the supernatant is carefully removed and discarded.

SECTION: Generation of cDNA library
43.  . This process is repeated twice. The samples are air-dried for 5 min  at Room temperature. Do not overdry the beads.

SECTION: Generation of cDNA library
44. 50 µL 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 2 min  at Room temperature.

SECTION: Generation of cDNA library
45. Next, 45 µL  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 5 min  at Room temperature.

SECTION: Generation of cDNA library
46. The samples are placed on a magnetic stand to separate the beads.
SECTION: Generation of cDNA library
47. After 5 min , the supernatant is carefully removed.

SECTION: Generation of cDNA library
48. Then, 200 µL  of 80% freshly prepared ethanol is added to the tube to wash the beads. After 30 s of incubation at Room temperature ,  . This process is repeated twice.

SECTION: Generation of cDNA library
49. The beads containing cDNA are air-dried for 5 min  at Room temperature. Do not overdry the beads.

SECTION: Generation of cDNA library
50. cDNA is eluted from the beads by adding 33 µL  of 1X TE. Mix well by pipetting up and down at least 10 times. The sample is incubated for at least 2 min  at Room temperature.

SECTION: Generation of cDNA library
51. The sample is placed on the magnetic stand. After 5 min  of incubation at Room temperature, 30 µL  of the solution is transferred to a new tube.

SECTION: Generation of cDNA library
52. 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.
SECTION: Generation of cDNA library
53. 40 ng  of cDNA is used for Illumina NGS (Next Generation Sequencing) library preparation.

SECTION: Generation of cDNA library
54. 40 ng of cDNA in 1X TE is mixed with 7 µL  of NEBNext Ultra II FS Reaction Buffer and 2 µL  of NEBNext Ultra II FS Enzyme Mix in a PCR tube. The final volume of the mixture is brought to 35 µL, and the sample is vortexed for 5 s.

SECTION: Generation of cDNA library
55. In a thermocycler, with the heated lid set to 75 °C, the following program is performed: 25 min at 37 °C and 30 min at 65 °C.


SECTION: Generation of cDNA library
56. 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. 
SECTION: Generation of cDNA library
57. The following components should be added directly to the above sample (35 µL). The adaptor should be added separately to each sample (DO NOT premix with ligation master mix and enhancer).


 
  

SECTION: Generation of cDNA library
58. 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 15 min  in a thermocycler without the heated lid.

SECTION: Generation of cDNA library
59. 3 µL  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 15 min .

SECTION: Generation of cDNA library
60. For the next step, the NEBNext Bead Reconstitution Buffer and the SPRI beads should be warm to Room temperature  for at least 30 min  before use.
SECTION: Generation of cDNA library
61. 57 µL  of SPRI beads are added to the PCR reaction. The sample is incubated for at least 5 min  at Room temperature. 

SECTION: Generation of cDNA library
62. The sample is placed on a magnetic stand.
SECTION: Generation of cDNA library
63. After 5 min  incubation, the supernatant is removed. Then, 200 µL of 80% freshly prepared ethanol is added to the tube. After incubation at Room temperature for 30 s, the supernatant is removed.  . This process is repeated twice.

SECTION: Generation of cDNA library
64. The beads containing cDNA are air-dried for 5 min  at Room temperature . Do not overdry the beads.

SECTION: Generation of cDNA library
65. 17 µL  of 0.1X TE is added to resuspend the beads. The cDNA-bead mixture is incubated for at least 2 min at Room temperature.

SECTION: Generation of cDNA library
66. The sample is placed on a magnetic stand. After 5 min, 15 µL of the cleared solution is transferred to a new PCR tube. 

SECTION: Generation of cDNA library
67. The following components are combined into a new PCR tube.


 

SECTION: Generation of cDNA library
68. Labelling with dual barcodes is performed by using the following PCR cycling conditions.


 

SECTION: Generation of cDNA library
69. For the next step, the NEBNext Bead Reconstitution Buffer and the SPRI beads should be warmed to Room temperature  for at least 30 min  before use.
SECTION: Generation of cDNA library
70. The PCR mixture is resuspended in 45 µL of SPRI beads. The sample is incubated for at least 5 minat Room temperature.

SECTION: Generation of cDNA library
71. The cDNA-bead mixture is placed on a magnetic stand to separate the beads from the supernatant.  
SECTION: Generation of cDNA library
72. After 5 min, the supernatant is removed and discarded.

SECTION: Generation of cDNA library
73. 200 µL  of 80% freshly prepared ethanol are added to the tube in the magnetic stand.  . This process is repeated twice.

SECTION: Generation of cDNA library
74. The beads containing cDNA are air-dried on a magnetic stand for 5 min  at Room temperature.

SECTION: Generation of cDNA library
75. The cDNA library is eluted by adding 33 µL  of 0.1X TE. Mix well by pipetting up and down 10 times. 

SECTION: Generation of cDNA library
76. The sample is placed on a magnetic stand. After 5 min,  30 µL  of the sample containing the cDNA library is transferred to a new tube. Libraries can be stored at -20 °C. 

SECTION: Generation of cDNA library
77. 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.