Anti-ORF1 IP with senescent cells
Date: 05/24/21 and 06/14/21
Previous experiment: May 24, 2021 - Anti-ORF1 IP with senescent cells
11 samples total (un-aged, 2 months, and 4 months)
• P15 (x3) 2.5x10^6 (processed on 05/24/21; 2 for MS and 1 for RIP seq)
• P17 (x4) (processed on 06/14/21; 2 for MS and 2 for RIP seq)
• 2 Mos (x5) 2x10^6 (3x processed on 05/24/21; and 2x processed on 06/14/21; 3 for MS and 2 for RIP seq)
• 4 Mos (x3) 2x10^6 (processed on 05/24/21; 2 for MS and 1 for RIP seq)
• N2102Ep control 50mg (x2; processed on 06/14/21, 1 for MS and 1 for RIP seq)
Scale: Treat each sample as 50mg IP
Extraction buffer: 20mM HEPES, pH7.4, 500mM NaCl 1% Triton X-100 with protease inhibitors; RNAse free L1 extraction buffer. 1:250 RNasin in EB, 1:1000 in wash.
Add 200ul of extraction buffer to each tube
Sonicate, 2 Amp for 4 x 1 sec
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Save 5ul of clarified lysate for Bradford Assay to determine how much material was actually used for IP
Use the rest of the lysate to set up IP with 10ul of anti-ORF1 beads per reaction
IP @ 4°C for 30’
After IP collect the flowthrough from all samples and save them in -80°C in case they could be useful
Wash beads 3x 1ml of extraction buffer
Transfer beads to fresh tubes during 2nd wash
The following samples were eluted in SDS (for MS)
4x early passage (2x P15: 05/24/21; 2xP17: 06/14/21)
5x senescent (2x 2mos: 05/24/21; 1x 2mos: 06/14/21; 2x 4mos: 05/24/21 )
1x N2102Ep control (06/14/21)
• Elute with 10ul of 2% SDS/40mM Tris, pH8 @ 70°C for 5’
• Collect the elution; snap frozen in LN2 and store in -80°C
• Prepare samples for MS using ultra high recovery S-trap protocol
MS sample preparation using S-Trap Micro Ultra-High Recovery protocol rev. 05/27/21
The following samples eluted in Trizol (for RNA-seq)
3x early passage (1x P15: 05/24/21; 2xP17: 06/14/21)
3x senescent (1x 2mos: 05/24/21; 1x 2mos: 06/14/21; 1x 4mos: 05/24/21)
1x N2102Ep control (06/14/21)
• Elute with 250ul of Trizol; vortex @ RT for 1’
• Collect the elution; snap frozen in LN2 and store in -80°C
• Prepare samples for RNA-seq using Direct-zol RNA MicroPrep kit (Zymo Research, Cat# R2062).
RNA extraction after cell powder IP with on column DNase treatment
• Check RNA quality by Bioanalyzer and send samples to John S. (1x P15, 2xP17, 2x 2mos and 1x 4mos)
RNA extraction with Zymo Direct-zol RNA MicroPrep kit
- 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 (“E”, pulse spin, place on magnet) 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, 4C (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 (20-30C) 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. Keep the remainder of samples in -80C freezer.
Bioanalyzer Pico chip loading order
1) P15
2) P17-1
3) P17-2
4) 2 mos-1
5) 2 mos-2
6) 4 mos
7) N2102Ep
First run of bioanalyzer result: RNA concentration of senescent samples was too low. N2102Ep concentration was too high (>15,000pg/ul), way above pico chip detecting level (50-5000pg/ul).
Bioanalyzer result https://drive.google.com/file/d/1NbApC3153uBeaSM-tJfv_7Q8DETG03Uw/view?usp=sharing
I ran the pico chip again. Made 1:4 dilution of N2102 sample and loaded it on the same pico chip. Ignore well 7 for this run. I messed it up. I loaded 1ul of diluted sample in well 7 by mistake and tried to take 1ul out because the concentration was already too high. Well 8 was the right control of diluted N2102Ep.
Bioanalyzer result https://drive.google.com/file/d/1_oWXf8vcYyz3in5z0vgVD6ROdG5a1ZNF/view?usp=sharing
Anti-ORF1 Western
Check anti-ORF1p signal in cell extract (“Input) (5ul saved and 2ul used to measure protein concentration) and supernatant after anti-ORF1 IP (“FT”) and SDS elution
Input and FT: 4ul of each (combined from two replicates of MS samples under each condition 2x 2ul=4ul). The samples are not normalized to 25ug each, but use 4ul out of ~200ul of cell extract (equivalent to 2% of the starting material for IP). I lost the input and FT for 4mos.
Elution: take 5% from two replicates of MS samples under each condition and combined (equivalent to ~10% of 50mg IP)
Run 15-well 4-12% Bis-Tris gel
1) Marker
2) Input_P15
3) FT_P15
4) Input_P17
5) FT_P17
6) Input_2mos
7) FT_2mos
8) Input_N2102EP (positive control)
9) FT_N2102EP (positive control)
10) Marker (not shown)
11) E_P15
12) E_P17
13) E_2mos
14) E_4mos
15) E_N2102EP (positive control)
Wet transfer, 70V for 1.5hr
Block both blots with TBST/5% milk @ RT for 1hr
Primary Ab: Mouse anti-ORF1 antibody, 1:1,000, RT for 2hr (2ug/ml)
3 quick rinse with TBST followed by wash with TBST, 3x 10’ @ RT
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
3 quick rinse with TBST followed by wash with TBST, 3x 10’ @ RT
Apply HRP substrate (Immobilon Fort Western HRP Substrate, Millipore, cat # WBLUF0100), RT for 1’
Develop the blot
30 second exposure, high sensitivity, auto tone
