Anti-ORF1 IP with senescent cells
Date: 05/24/21 and 06/02/21
11 samples total (un-aged, 2 months, and 4 months)
• P15 (x3) 2.5x10^6 (done on 05/24/21; 2 for MS and 1 for RIP seq)
• P17 (x4) (done on 06/02/21; 2 for MS and 12 for RIP seq)
• 2 Mos (x5) 2x10^6 (3x done 05/24/21; and 2x done on 06/02/21; 3 for MS and 2 for RIP seq)
• 4 Mos (x3) 2x10^6 (done on 05/24/21; 2 for MS and 1 for RIP seq)
Scale: Treat each sample as 50mg IP (3 replicates from each set)
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 (We will skip Western this time. But the Bradford Assay will give us an idea how much initial material we are dealing with. This could be a useful information for future experiments).
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
2 replicates will be eluted in SDS (for MS or DNA work)
• 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. 12/22/20
1 replicate will be eluted in Trizol (for RNA-seq)
• 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 or send samples to John S.