Effects of ZnCl2 and hUdT on IP specificity and RNA quality
Date: 5/10/21
Cell line and CRC tissue: N2102Ep and 174T
Extraction/Wash buffer conditions
20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 with protease inhibitors and with RNasin and ZnCl2 plus new RNasin inhibitor (hUdT) at different concentrations and protease inhibitors
RNasin: for N2102Ep, 1:250 in extraction buffer and 1:1000 in wash; for 174T, 1:40 in extraction buffer and 1:250 in wash
Protease inhibitors 1x in all buffers
Scale: 50mg scale with 2.5, 5 or 10ul of anti-ORF1 per reaction
Weigh out 5x 50mg of N2102Ep and 4x 50mg of 174T
Add 200ul 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)
Set up 10x 50mg IP with snit-ORF1 beads
1. N2102Ep_anti-ORF1_RNasin (1:250) + 25mM EDTA_5ul beads
2. N2102Ep_anti-ORF1_RNasin (1:250) + 1mM EDTA_5ul beads
3. N2102Ep_anti-ORF1_1mM ZnCl2 + 200uM hUdT_5ul beads
4. N2102Ep_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_5ul beads
5. N2102Ep_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_10ul beads
6. 174T_anti-ORF1_RNasin (1:40) + 25mM EDTA_5ul beads
7. 174T_anti-ORF1_RNasin (1:40) + 1mM EDTA_5ul beads
8. 174T_anti-ORF1_1mM ZnCl2 + 200uM hUdT_5ul beads
9. 174T_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_5ul beads
10.174T_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_2.5ul beads
IP @ 4°C for 30’
During IP, check protein concentration of clarified lysate by Bradford assay
Wash 2x 250ul of extraction buffer
Switch beads to fresh tubes at 2nd wash step and at the same time split beads into two equal aliquots (50% for Western and 50% for RNA prep)
a) 50% of beads: add 10ul 1.1x LDS, 70°C for 5’; collect the elution for Western b) 50% of beads: elute in 250ul of Trizol (follow the steps of RNA extraction below)
Western
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_RNasin (1:250) + 25mM EDTA_5ul beads
3) N2102Ep_anti-ORF1_RNasin (1:250) + 1mM EDTA_5ul beads
4) N2102Ep_anti-ORF1_1mM ZnCl2 + 200uM hUdT_5ul beads
5) N2102Ep_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_5ul beads
6) N2102Ep_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_10ul beads
7) 174T_anti-ORF1_RNasin (1:40) + 25mM EDTA_5ul beads
8) 174T_anti-ORF1_RNasin (1:40) + 1mM EDTA_5ul beads
9) 174T_anti-ORF1_1mM ZnCl2 + 200uM hUdT_5ul beads
10) 174T_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_5ul beads
11) 174T_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_2.5ul beads
12) Space
13) MT302 anti-ORF1 (5% of 100mg IP)
Wet transfer 70V for 1.5h
Blocking: TBST/5% milk, RT 2hr
Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C overnight
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
10 second exposure, high sensitivity, auto tone
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/1bhF9Pa0eEzA3ldO7ktMonLCPiUVQgkur/view?usp=sharing
10 samples total (samples 6-10 RNA concentration was too low, will repeat RNA prep. see May 13, 2021 - Effect of ZnCl2 and hUdT on CRC RNA quality
1. N2102Ep_anti-ORF1_RNasin (1:250) + 25mM EDTA_5ul beads
2. N2102Ep_anti-ORF1_RNasin (1:250) + 1mM EDTA_5ul beads
3. N2102Ep_anti-ORF1_1mM ZnCl2 + 200uM hUdT_5ul beads
4. N2102Ep_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_5ul beads
5. N2102Ep_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_10ul beads
6. 174T_anti-ORF1_RNasin (1:40) + 25mM EDTA_5ul beads
7. 174T_anti-ORF1_RNasin (1:40) + 1mM EDTA_5ul beads
8. 174T_anti-ORF1_1mM ZnCl2 + 200uM hUdT_5ul beads
9. 174T_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_5ul beads
10.174T_anti-ORF1_0.1mM ZnCl2 + 200uM hUdT_2.5ul beads