Isolation of mRNA undergoing translation in the RiboTag model (Ribosomal HA tag) from the mouse heart, brain and lung

Isolation of mRNA undergoing translation in the RiboTag model (Ribosomal HA tag)

Buffers and Reagents

Homogenization buffer (HB)

                                                          Stock solution           per 10 ml

50 mM Tris (pH 7.4)                        1M                                  500 µl
100 mM KCl                                     1M                                  1000 µl
12 mM MgCl2                                  1M                                  120 µl
1% NP40                                         5%                                  2000 µl
DEPC H2O                                                                             6380 µl

Supplemented homogenization buffer (HBS)

                                                            Stock solution         per 10 ml

1 mM DTT                                          1M                               10 µl
Protease inhibitors                                                                  1 tab
200 U/ml RNasin (or RNAseOut)       40 U/µl                         50 µl
100 µg/ml cycloheximide                    10 mg/ml                     100 µl
1 mg/ml heparin                                  100 mg/ml                   100 µl
Homogenization buffer                                                           9740 µl

High salt buffer

                                                            Stock solution         per 10 ml

50 mM Tris (pH 7.4)                           1M                               500 µl
300 mM KCl                                        1M                               3000 µl
12 mM MgCl                                       1M                               120 µl
1% NP40                                             5%                               2000 µl
1 mM DTT                                          1M                               10 µl
100 µg/ml cycloheximide                    10 mg/ml                     100 µl

100 U/ml RNAsin (or RNAseOut)      40 U/µl                         25 µl
DEPC H2O                                                                             4235 µl

Notes:

·         Cycloheximide (Sigma, Cat# 01810).

·         make cyclohexemide: 10mg/ml, weight 50mg cyclohexamide to 5ml RNase free water.  Vortex to dissolve. Solution can be kept at 4C for up to 2 weeks.

·         Homogenization buffer can be made and stored at 4°C for a month or so, the supplemented buffer and the wash buffer can’t be stored longer than a day at 4°C because of the cycloheximide.

·         NP40: Igepal CA-630 (Sigma, Cat# 56741).

·         Protease inhibitors: Complete Mini EDTA-free (Roche Cat# 11836170001).

·         Heparin sodium salt (Sigma, Cat# H4784 -250mg).

·         Pierce™ Protein A/G Magnetic Beads (Thermo Fisher Scientific, Cat# 88803).

·         RNA isolation use RNeasy Plus mini kit (Qiagen, Cat# 74134).

Harvest tissue
Perfuse mice with 12 ml cycloheximide (100 µg/ml in PBS) before harvesting tissues for about 2 minutes. Freshly extract tissues from mice. Snap freeze tissues in liquid N₂ and store at -80°C until processing (processing should be as soon as possible to reduce degradation, do not delay more than two weeks).

Lysate preparation

1.    Use cold supplemented homogenization (HBS) buffer to homogenize the tissue. Grind the brain tissue with Dounce homogenizer using 40 strokes of the loose-fitting pestle (type A pestle) and additional 40 more strokes with the tight-fitting pestle (type B pestle). Grind the lung and heart tissue by FastPrep-24™ 5G benchtop homogenizer (MP Biomedicals, Model:116005500) with lysis beads (MP Biomedicals, Cat# 116938005 - 5 x 15 ml) at 4^oC. Set the speed at 6 to 8, for 15 second and 3 repeat. Keep the tissue on ice for 5 minutes between breaks to maintain low temperature. Based on tissue weight, add the remainder of HBS + to make a final w/v ratio=5%.

2.    After homogenization, transfer lysate to a microcentrifuge tube and continue incubation on ice for 15 minutes.

3.    Clear the lysate by centrifuging 15 min at 10,000 × g, 4°C. Transfer the supernatant to new microcentrifuge tubes. Discard the resulting pellet.

4.    Centrifuge samples again for 10 min, 10,000 × g, 4°C. Transfer the supernatant to new microcentrifuge tubes. Discard resulting pellet.

5.    Inspect the samples, remove any residual fatty/tissue residues if necessary.

6.    Remove a small aliquot of the cleared lysate (40 μl) and store it at −80°C for subsequent Input analysis (RNA extraction).

Immunoprecipitation

7.    Add 3 μl of CHIP graded rabbit hemagglutinin antibody (Abcam polyclonal, Cat# ab9110) to 400 μl of the remaining cleared lysate and incubate for 1 hr at 4°C on a microtube rotator with gentle mixing (end-over-end). 
For the IgG control, use 3 μl (=1µg) of mouse monoclonal IgG1 antibody (Sigma, Cat# M5284).

8.    Immediately prepare the magnetic protein A/G beads (100µl/sample): Take 100 μl of protein A/G beads, remove the stocking buffer, add 800 μl of homogenization buffer, rotate in 4^oC for 30 min. Place the tube into the magnetic stand on ice to collect the beads and remove and discard the storage buffer. 
Add 400 μl of homogenization buffer and rotate end-over-end for 15 min to wash the beads twice.

9.    At 1 hour incubation, remove homogenization buffer from the beads, transfer the 400 μl sample+Ab into the beads. Incubate samples for 1 hour at 4°C in a tube rotator with gentle end-over-end mixing.

10.  Place incubation samples into the magnetic stand on ice and remove supernatant. 


11.  With the tubes on the stand, add 400 μl of high-salt buffer to remove nonspecific binding from the immunoprecipitate (IP). Transfer tubes to the rotator and wash the IP with gentle end-over-end mixing for 5 min at 4°C.

12.  Repeat high-salt washes two more times as described in step 10, for a total of three times.

13.  Re-suspend beads in 800 μl of high-salt buffer, mix on rotator for 5 min. Transfer beads and buffer to a clean tube. This step helps remove any RNA or polyribosomes that might be sticking to the original tube.

14.  Place tubes on the magnetic stand for at least 1 min, then carefully remove all high-salt buffer.  add 350 µl RLT Plus Buffer (+ß mercaptoethanol 10 μl/ml) to the beads and vortex for 30 seconds. Transfer the RLT Plus buffer from beads which now includes the RNA to new tube. Buffer RLT plus is from RNeasy plus Mini kit (Qiagen).

RNA extraction

15.  Tissue lysates were spun through gDNA Eliminator spin columns from RNeasy plus Mini kit (Qiagen) to remove genomic DNA.  RNA was extracted and eluted in 40 μl H₂O for RNA qualification and sequencing.