Nucleic acids characterization in L1 RNPs_Collaboration with John Sedivy
Date: 02/16/21
Cell lines: N2102Ep
Scale: 100mg; Powder to beads ratio: 100mg per IP reaction with 20ul of anti-ORF1 or anti-ORF2 beads
Everything should be done with RNA-grade, clean buffers and new boxes of tips and tubes etc whenever reasonably possible.
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors only in the extraction buffer; add RNasin @ 1:250 in extraction buffer and 1:1000 in wash buffer)
Anti-ORF1 and anti-ORF2 IPs
Weigh 6x 200mg of N2102Ep
Add 800ul of extraction buffer (1:4 w/v), vortex to mix
Sonicate 4 x 2 sec at 2 Amp; repeat once if necessary (total energy output should be ~30J)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Combine clarified lysate from all 6 tubes (6x 200mg cell extract)
Evenly split it into 12 aliquots (for 12x 100mg IP reactions)
Set up 100mg IP reactions (triplicates for each)
1) N2102Ep_anti-ORF1 (anti-ORF1: mouse antibody)
2) N2102Ep_mIgG (beads coupled with mouse IgG)
3) N2102Ep_anti-ORF2 (anti-ORF2: rabbit antibody)
4) N2102Ep_rIgG (beads coupled with rabbit IgG)
Incubate @ 4°C for 30’
After IP, wash 3x 1ml extraction buffer
Transfer the beads to fresh tubes during the 2nd wash
Proteinase K digestion
Proteinase K (ThermoFisher Scientific, Catalog number: 25530049): 20mg/ml; treat this as 100x (final concentration in the digestion mix should be 200ug/ml)
Proteinase K digestion buffer: 0.5% w/v SDS, 40mM Tris pH 8.0, 200mM NaCl, 1mM EDTA
Reaction volume: 30ul per tube; 12 reactions (360ul total)
Making Proteinase K digestion mix by adding 4ul of 20mg/ml Proteinase K to 400ul of Proteinase K digestion buffer (1: 100 dilution); mix completely by vortexing and quick spin
Add 30ul of digestion mix to each tube
Incubate @ 37°C for 30’ with mixing
After digestion, transfer the digestion mix to another set of tubes and keep on ice SDS elution
Elution buffer: 2% w/v SDS, 40mM Tris pH 8.0, 200mM NaCl, 1mM EDTA
Add 20ul of elution buffer to one tube, vortex, spin down, remove, combine with 30ul of matching digestion mix previously collected from the same tube (50ul total: digestion mix + eluate)
Handle each set of 3 replicates at the same time (4 sets total)
Final samples to be sent
Snap-freeze samples and keep them at -80°C (3 replicates of each condition, 12 total) before sending out
12 samples total:
• N2102Ep anti-ORF1 (1-3)
• N2102Ep mIgG (1-3)
• N2102Ep anti-ORF2 (1-3)
• N2102Ep rIgG (1-3)
Results from Sedivy lab
https://drive.google.com/file/d/1GMMOkKvsmz81db0Ll2jl-4zGi8Kw2uQO/view?usp=sharing