N2102Ep anti-ORF1 IP with or without hUdT with IgG control
Date: 4/30/21
Everything should be done RNA-grade with nuclease-free reagents, clean bench and pipets with RNase Zap; Use newly prepared buffers and new boxes of tips and tubes etc. whenever reasonably possible. Change gloves often!
RNasin: Recombinant RNasin® Ribonuclease Inhibitor (Promega, Cat # N2515)
hUdT: 3'-N-hydroxyurea-3'- deoxythymidine 5'-monophosphate, in free form (from Ali Alipour Najmi Iranag, University of Groningen). MW: 380.25g/mole (1M->380.25g/L)
Direct-zol RNA MicroPrep (Zymo Research, Cat # R2060)
Extraction/Wash buffers (both with inhibitors):
Extraction/Wash buffers were made with Nuclease-free water (the stock solutions are nuclease free) with Protease inhibitors used at 1x during extraction and washes
1) 20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 (no RNase inhibitors)
2) 20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 with 1mM ZnCl2 and 200um hUdT
3) 20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 with 1mM ZnCl2 and 200um hUdT + RNasin (1:250 in extraction buffer and 1:1000 in wash)
Cell line: N2102Ep
Scale: 100mg scale with 20ul anti-ORF1 beads or 20ul of mouse IgG beads per reaction (4 conditions in duplicates; 8 samples total)
Weigh out 8x 100mg of N2102Ep
Add 400ul of extraction buffer with protease inhibitors and RNase inhibitors to each tube according to the IP conditions below
Resuspend the powder by vortexing
Sonicate @ 2 Amp; 4x 2sec (15-20J)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
During spin, wash 160ul anti-ORF1 beads with extraction buffer
Set up 8x 100mg IP with 20ul of anti-ORF1 beads per 100mg reaction
1-2: N2102Ep_anti-ORF1_no inhibitor
3-4: N2102Ep_anti-ORF1_Zn+hUdT
5-6: N2102Ep_mIgG_Zn+hUdT
7-8: N2102Ep_anti-ORF1_Zn+hUdT+RNasin
IP @ 4°C for 30’ (get tubes and Zymo columns ready while IP is going)
Wash 2x 500ul of extraction buffer
Prepare two sets of new tubes
Split beads in each tube into 2 equal aliquots (each equivalent to 50mg IP) and transfer them to new sets of tubes; one set for gel (elute in 10ul 1x LDS) and one set for RNA prep (elute in 250ul of Trizol)
Switch beads to fresh tubes at 2nd wash step
LDS elution: 10ul 1.1x LDS, 70°C for 10’; collect the elution for gel (Coomassie stain)
Trizol Elution: Elute in 250ul of Trizol (follow the steps of RNA extraction below)
Protein Gel
Add 1ul of 500mM DTT to each sample
Heat @ 70°C for 10’
Run 4-12% Bis-Tris gel
1) Marker
2) N2102Ep_anti-ORF1_no RNase inhibitor_1a
3) N2102Ep_ anti-ORF1_no RNase inhibitor_1b
4) Space
5) N2102Ep_ anti-ORF1_Zn_hUdT_2a
6) N2102Ep_ anti-ORF1_Zn_hUdT_2b
7) Space
8) N2102Ep_ mIgG_Zn_hUdT_2c
9) N2102Ep_ mIgG_Zn_hUdT_2d
10) Space
11) N2102Ep_ anti-ORF1_Zn_hUdT_RNasin_3a
12) N2102Ep_ anti-ORF1_Zn_hUdT_RNasin_3b
13) BSA_50ng
14) BSA_150ng
RNA extraction
- all steps from here may be performed at RT if not mentioned otherwise
Vortex for 1min, place the tubes aside (can be on ice or RT, Trizol extract is stable at RT)
Spin the Phasemaker tube at 16k RCF, 30 sec
Add 50ul of chloroform and 25ul H2O to the Phasemaker tube do this immediately before adding elutions or (perhaps safer still) immediately after transferring elutions to the Phasemaker tube. Adding chloroform / H2O too far in advance may cause a failure. not adding the water will cause the Phasemaker tube to fail and 'swallow' the aqueous portion of the Trizol reagent mix.
Collect the elution and transfer to the Phasemaker tube
Mix by hand, vigorously for 15 sec
Incubate 2 min @ RT with end-over-end mixing this step is almost certainly dispensable for IP - it is supposed to be time for RNPs to dissociate.
Spin 16k RCF, 4°C (not important, Trizol extract is stable at RT), 5 min
Transfer the aqueous phase to a new tube by pressing pipette tip against wall of tube; avoid puncturing the Phasemaker gel
Add an equal volume of 100% EtOH (~185uL) to each sample and mix thoroughly, vortex and pulse spin
Transfer the mixture to a Zymo-Spin IC column in a collection tube and centrifuge @ 16,000rcf for 30 seconds
DNase I treatment
• Add 400ul RNA Wash Buffer to the column and centrifuge @ 16,000rcf for 30 sec
• In an RNase-free tube, add 5ul DNase I 95U/ul), 35ul DNA Digestion Buffer and mix. Add the mix directly to the column
• Incubate @ RT (20-30°C) for 15 minutes
Add 400ul Direct-zol RNA PreWash to the column and centrifuge @ 16,000rcf for 30 seconds.
Discard the FT and repeat this step. Use the vacuum-trap system to clear the liquid from the tubes both times
Add 700ul RNA Wash Buffer to the column and centrifuge for 2 minutes @ 16,000rcf to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.
To elute RNA, add 6ul of RNase-Free Water directly to the column matrix and centrifuge @ 16,000rcf for 30 seconds.
Send 1.5ul of Elution for Pico chip bioanalyzer analysis. save the rest in -80°C freezer
Bioanalyzer result
https://drive.google.com/file/d/1KsmJFB860AGX8I2pJ8tx5CG9Up6Ip17Z/view?usp=sharing