Materials for CRC tissues RNA-seq and TIP-seq
John LaCava <jlacava@mail.rockefeller.edu> Tue 1/7/2020 8:57 AM To: Leila Saba <l.j.saba@umcg.nl> Cc: Hua Jiang <hjiang01@mail.rockefeller.edu>; John LaCava <jlacava@mail.rockefeller.edu>
About the amount to save - we can confirm how much based on how much we sent to Kathy Burns last time to do TIP-seq. We will want to send her these materials also - or, at least have the materials in case we need to do it ourselves.
I would like to do TIP-seq on intact and milled material to see if the milling makes a difference.
We should start to assemble an overall procedural protocol for all manipulations that will be applied to CRCs.
Saba, LJ (eriba) <l.j.saba@umcg.nl> Tue 1/7/2020 8:10 AM To: 'Hua Jiang' <hjiang01@mail.rockefeller.edu> Cc: John LaCava <jlacava@mail.rockefeller.edu>
I just met with John and this is the update on the CRC IP project. Please mill the following tumors, first setting aside ~300mg of each tumor PRE-MILL for TIPseq:
Case#
174 Metastatic breast carcinoma
185 MMMT ovary
197 Adenocarcinoma sigmoid colon
198 Adenocarcinoma right colon
268 Ovarian carcinoma
284 Clear cell carcinoma ovary
Numbers above 200 should be in rack B3 of the newest -80C, numbers below 200 should be in the chest freezer from Bronk 409. These are in numerical order by case#, 268 is the one Marty scored highest for Orf1 status. I am emailing Marty separately about the other case #s he recommended that I don’t think we have at RU. So there might be additional tumors to add to the list later, but please start with these for now.
We will also definitely be continuing with the three 162 Liver Mets that are already milled, and possibly 159, but again I will have to update you on the latter after checking with Marty.
Update on tumor tissues (Hua June 8, 2020)
174 Metastatic breast carcinoma (still could not find this one)
185 MMMT ovary: VDE1858 Ovary mmmt, 4 full Falcon tubes of Tumor, No Normal
197 Adenocarcinoma sigmoid colon: 1 Falcon tube of Tumor (~7g) and 1 Falcon tube of Normal (~7g)
198 Adenocarcinoma right colon: 1 Falcon tube of Tumor (~7g) and 1 Falcon tube of Normal (~5g)
268 Ovarian carcinoma: 2 Falcon tubes: 1 black labeled “P1858 268 Normal spleen”, 1 purple labeled “P1858 268 #268” (tumor?), ~30g each
284 Clear cell carcinoma ovary: 2 Falcon: both labeled “P1858 284”, ~40g each (tumor, no normal?)
Previous experiments of tissue RNA preps
Saba, LJ (eriba) <l.j.saba@umcg.nl> Sun 6/7/2020 9:50 AM To: 'Hua Jiang' <hjiang01@mail.rockefeller.edu>; John LaCava <jlacava@mail.rockefeller.edu>
This is the best protocol we have, I did not see anything with better results by Lars or Angelica: 23/01/2019_162T RNA Preparation - Zymo and Phaselock We switched to the phaselock and zymo combo because it is easier and faster than the glycoblue + precipitation. Our results with MT302 were just as good or better, see lane 8 on the pico chip run of above experiment. RNasin there is 1:40 but I believe we went as low as 1:80 with similar results. When I initially did the RNA IP optimization experiments in tumor, we saw that sonication did not make a difference in RNA quality, but I never went back and checked protein quality/yield, although I definitely saved some protein samples for that purpose. I don’t know if it is worth taking a look at them now, after over a year at -20. See here, lane 3 is no sonication. 08/01/2018_Extract RNA from Human Tissues (optimize tissue RNA prep conditions). (We ultimately concluded that superasin was not doing anything in our samples)
Leila’s old experiment
23/01/2019_162T RNA Preparation - Zymo and Phaselock
Extraction/Wash buffer:
20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 Extraction/Wash buffer:
1. made with Nuclease-free water (the stock solutions are nuclease free) with 2. protease inhibitors used at 1x during extraction and washes 3. RNasin used at 1:40 each in extraction buffer and 1:250 during washes
• All tumor lines: sonicate 4x1 sec at 2 Amp (energy output ~10J) • MT302: Sonicate for 5x2 sec at 2 Amp; (energy output ~20J) (had to repeat 1x) • IP@ 4°C for 30’
Hua’s old experiment
09/09/19 - RNVC Testing – RNA Prep from MT302 and 162T
Best condition seems to be:
5) 162TA 1:40 RNasin EB; 1:250 RNasin wash
• Resuspend the powder by vortexing, no sonication
• IP @ 4°C for 5’
Optimization of tumor IP conditions
Hua’s old experiment
05/28/19 - 162T and 162N anti-ORF1 IP_Sonication_Beads titration_Time course
Conclusion: no sonication (-sonication), IP for 5’ is fine at protein level
Update on CRC tissues
