So, it turns out my 6 pooled samples aren’t concentrated enough for NWGC to sequence. They are all at around 25ng/ul, but they need to be at least 50ng/ul. I called zymo yesterday to find out how best to do that. They recommended a kit (free sample coming next week). With Sam’s guidance, I tried using the Microprep Kit columns and washes to concentrate some pools that I made today out of extra extracted RNA. It kinda worked, I think… but there was some loss of RNA, so I’m worried about using that method with my pooled 6 samples. Details in the post.
I created the following pools:
- tubes: 325, 303, 321, 301
- tubes: 343, 312, 357, 329
I didn’t think to run the pooled samples on the qubit before trying to concentrate them, so the results don’t mean anything. They can be found in the comments of this github issue, though: #798
1. Create pools
- Pool 3 –> tubes: 310, 315, 341, 337 (41ul of pooled sample)
- Pool 4 –> tubes: 201, 203, 220, 236 (40ul of pooled sample)
2. Qubit 2ul of pooled samples RNA HS Kit so I know the baseline RNA concentration
- Pool 3 –> 2ul on qubit: 25.2 ng/ul. Total RNA in remaining sample: 982.8 ng of RNA.
- Pool 4 –> 2ul on qubit: 16.4 ng/ul. Total RNA in remaining sample: 632.2 ng of RNA.
3. Concentrate samples
- Used the Zymo Quick-DNA/RNA Microprep Plus Kit and started at Step 2 on page 7.
- Eluted RNA in 35ul of TE (NWGC requires eluted RNA to be in at least 30ul of TE)
4. Run 2 ul of concentrated RNA samples on qubit RNA HS Kit
- Pool 3 –> 2ul on qubit: 23.7 ng/ul. Total RNA in remaining sample: 782.1 ng of RNA.
- Pool 4 –> 2ul on qubit: 14.1 ng/ul. Total RNA in remaining sample: 465.3 ng of RNA.
There is some loss of RNA in the process, and I’m worried about doing this with my pooled samples, becuase they are all around 1100ng of total RNA, and NWGC requires at least 1000ng of RNA.
I’m wondering if maybe I should add a few more samples of RNA to the 6 pools, and then try what I did today, or if I should just look into the other options I list below.
- Free sample of the Zymo RNA Clean Concentrator 5 R1013 will hopefully arrive on Tuesday, so I can try to use that and get the samples in on Tuesday (I leave for the winter break on Wednesday, Dec. 18th in the morning)
- See if what Shelly suggested for Yaamini’s samples might work for mine? (Yaamini’s notebook posts: WGCS Samples; WGS Samples Part 2)
In other news…
- Submitted abstract for a talk or poster to NSA today (didn’t specify one or the other, becuase i’d be kinda down to try making one of the better posters)
- Am working on an application for a SAFS fellowship for my Spring 2020 quarter so that I don’t have to TA while I’m finishing up my thesis and defending (will submit before I leave for CA next Wednesday)