BG4 RIP-seq using adult mouse forebrain tissues

1. Brain dissection and lysis

        i.      Collection of mouse forebrains

           a.   Euthanize mice (6 – 8-week-old) with cervical dislocation.

       b.  Decapitate and scalp the mouse.

       c.   Cut the skull from cerebellar side with scissors along with the longitudinal fissure.

       d.   Remove the skull with flat-tipped tweezers (peel outward on both sides).

       e.   Scoop up the cortices with a spatula of appropriate size.
       f.    Collect the forebrain portions and place them in a 50 mL conical tube filled with the cold brain collection buffer with RNase inhibitor.

        ii.     Lysis using Dounce homogenizer

a.  Transfer the forebrain tissues from three mouse brains at a time to a glass Dounce homogenizer containing 7.5 mL of the cold brain collection buffer with RNase inhibitor. 

b.  Gently homogenize the tissues with 10 strokes of the loose pestle, followed by 10 strokes of the tight pestle.

         c.  Transfer the homogenate to a conical tube.

d.  Centrifuge the conical tube at 1,000 g for 10 min at 4°C in a swinging bucket rotor to pellet cellular debris and nuclei.

e.  Take about 6 mL supernatant to a new conical tube (henceforth referred to as forebrain lysates).

2. RNA immunoprecipitation

    i.       Preclear

a.    Remove the storage solution of the Protein G HP SpinTrap column (containing Protein G Sepharose gels) by centrifugation for 1 min at 150 x g.

b.   Wash the Protein G HP SpinTrap column thrice with 400 μL of TBS (50 mM Tris, 150 mM NaCl, pH 7.5).

c.    Add 200 μL of TBS and mix briefly.

d.   Centrifuge at 150 x g for 1 min at 4°C and discard the supernatant.

e.  Wash the column once with 400 μL of the brain collection buffer with RNase inhibitor.

f.    Centrifuge at 150 x g for 1 min at 4°C.

g.   Discard the supernatant and add 400 μL of forebrain lysates (from step 1-ii-e).

h.   Incubate the tube with rotation for 1 h at 4°C.

    ii.      Antibody immobilization onto Sepharose gels

a.    Remove the storage solution of the Protein G HP SpinTrap column (containing Protein G Sepharose gels) by centrifugation for 1 min at 150 x g.

b.  Wash the Protein G HP SpinTrap column thrice with 400 μL of TBS.

c.    Add 200 μL of TBS containing 15 ug of BG4 & anti-6xHis antibody or normal IgG.

d.   Fully suspend by manual inversion.

e.    Incubate the tube with rotation for at least 30 min at 4°C.

f.    Centrifuge for 1 min at 150 × g to remove excessive antibody.

g.   Wash once with TBS and centrifuge at 150 x g for 1 min at 4°C.

    iii.      BG4 enrichment

            a.      Centrifuge the tube from step 2-i-h at 150 x g for 1 min at 4°C.

        b. Transfer the precleared cell lysates to the tube from step 2-ii-g.

        c. Incubate the tube with rotation for 3 hours at 4°C.
        d.   Wash with 400 μL of the Wash Buffer (provided with the RiboCluster Profiler RIP-Assay Kit) with 1.5 mM DTT 5 times.

e.    Centrifuge at 150 x g for 1 min at 4°C and discard the supernatant.

f.    Add 240 μL of the Master mix (Solution I : Solution II = 1:39, provided with RiboCluster Profiler RIP-Assay Kit) solution and vortex thoroughly.

g.   Add 150 μL of the Solution III (provided with the RiboCluster Profiler RIP-Assay Kit) and vortex thoroughly.

h.   Centrifuge at 1,000 × g for 1 min at 4°C.

i.    Repeat step f–h and combined the eluates.

    iv.      RNA isolation

Purify the combined eluates with the RNeasy Mini/Micro Kit (Qiagen) following the manufacturer's instructions.

3. rRNA-depletion using the RiboMinus™ Eukaryote System v2

        Follow the manufacturer's instruction.

4. Library preparation using the Ion Total RNA-Seq Kit v2

        Follow the manufacturer's instruction.

Materials

• Brain collection buffer with RNase inhibitor 

[20 mM tris-HCl containing 0.32 M sucrose, pH 7.5 and RNase inhibitor (1x)]

• TBS (50 mM Tris, 150 mM NaCl, pH 7.5)
• RiboCluster Profiler RIP-Assay Kit (Medical & Biological Laboratories)
• Protein G HP SpinTrap (GE Healthcare)
• BG4 (Ab00174-10.6, absolute antibody)
• Anti-6xHis antibody (ab18184, abcam)
• Normal rabbit IgG (provide with the RiboCluster Profiler RIP-Assay Kit)
• RNeasy Mini/Micro Kit (Qiagen)
• RiboMinus™ Eukaryote System v2 (Thermo Fisher Scientific)
• Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific)