RIME

RIME Protocol    by Ioana Nitulescu and Dylan Reid | December 2019

adapted from H. Mohammed et al., Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes. Nat Protoc 11, 316-326 (2016)

Day 1: Nuclei Isolation, Click reaction, Shearing

1. Make solutions:
   1. Roche 25x: 10 mL PBS + 5 pellets (Roche #05056489001).
   2. Lysis Buffer 1. 50 mM HEPES pH 7.4, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100. Add Roche to 1x before use.
   3. Lysis Buffer 2. 10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA. Add fresh Roche to 1x before use.
   4. RIME-RIPA buffer. 50 mM HEPES (pH 7.6), 1 mM EDTA, 0.7% (wt/vol) sodium deoxycholate, 1% (vol/vol) NP-40 and 0.5M LiCl. Add fresh Roche to 1x before use.
   5. TF Sonication Buffer. 300 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1% Na-Deoxycholate,0.1% SDS, 1% Triton X-100, 0.25% Sarkosyl. Add fresh Roche to 1x before use.
   6. Click reaction reagents from ThermoFisher Click-iT EdU kit (#C103370) and Biotin-TEG-azide (Barry & Associates #BT1085). Make master mix (10 mM CuSO4, 5 mM THPTA, 1 mM Biotin-TEG-Azide, 125 mM sodium ascorbate) right before use.
2. Thaw 4 200M crosslinked iN pellets labelled with EdU for 1 day (X-link conditions: 8 min, 1% formaldehyde + quenched with 1 mg/mL BSA in PBS) on ice and resuspend each in 40mL Lysis Buffer 1. 10 mL/50 M cells, so need 160 mL total LB1, with 40 mL/tube.
3. Rock on nutator for 10 min at 4°C. Spin at 4°C for 1,400xg for 5 minutes. Aspirate liquid.
4. Add Lysis Buffer 2 (40 mL per tube) then rock on nutator for 10min at 4°C. Make sure you dislodge the pellet, just do it gently without pipetting.
5. Spin at 4°C for 1,400xg for 5 minutes. Aspirate liquid.
6. Perform CuAAC Click reaction to generate biotin epitope for pull-down. Resuspend each sample in 2.7 mL CuAAC click reaction buffer (10 mM CuSO4, 5 mM THPTA, 1 mM Biotin-TEG-Azide, 125 mM sodium ascorbate) or PBS (no click control). Incubate at RT for 45 min in the dark.
7. Wash 3x in cold DPBS + Roche 1X.
8. Spin at 4°C for 1,400xg for 5 min. Aspirate liquid.
9. Resuspend nuclei samples in TF Sonication Buffer, 8 mL each sample, and divide among 4 5mL Eppendorf tubes. Total amount of tubes: 16 tubes. 32 mL buffer needed total.
10. Shear each separately for 4 min (Epishear Sonicator, pulse 1 s on, 1 s off, 25% amplitude) on cold block. 
11. Recombine the fractions of each sheared sample, spin down 16,000 x g for 10 min at 4°C.
12. Add 200uL Dynabeads Streptavidin to eppendorf tubes with 1 tube of beads per pulldown. Wash 1X with PBS. Remove SN with aspirator. 
13. Add samples to preblocked Streptavidin beads and incubate O/N on a rotator at 4°C. 

Day 2: Elution

All washes and buffers at 4°C. Chill magnetic stand before use.

1. Collect beads with magnetic stand. Aspirate SN. Wash with 1mL RIME-RIPA buffer 10x at 4°C. 
2. Wash the beads twice in 1mL of cold freshly made 100 mM ammonium hydrogen carbonate (AMBIC) solution and transfer beads to new tubes after the second wash. Spin down on tabletop minifuge right before apirating to collect the beads above the liquid line (otherwise they will be loose).
3. Snap freeze the beads and hand over to Mass Spec core for on-bead digest and downstream steps.

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