Pooling libraries

I’m ready to send my libraries out for sequencing! First, I need to determine my sample pools. Carolyn suggested creating pools of about 8 samples each that all have roughly the same concentration. This way, they can do QC and spike in more of the low volume samples if needed.

This means I need to create 11 pools. I sorted my library information by cDNA concentration and assigned pools that way. The information can be found here. I reviewed the list twice to ensure that each sample had a unique index, since I ordered the samples by combined indices to make it easier to pool the samples (much easier to figure out which pool a in a strip goes to, as opposed to a mad hunt for all the libraries that should go into one pool). A few notes:

• To actually pool my samples, Carolyn suggested I add ~10 µL of the library to each pool. I added 10 µL of library to each pool, except for 5-135. For some reason, I only had 10 µL of this sample! I added 5 µL of pool. I’m hoping that this is because it maybe got less TE so it’s more concentrated, and not because I didn’t transfer all the eluted library.
• Prior to adding the aliquot to a pool, I took the strip tube and flicked it, then spun it down. I forgot to flick mix for samples 15-161, 15-092, 5-009, and 25-202. By the time I realized I forgot to mix the samples, I decided to pipet mix 25-202, 5-004, 5-121, and 5-069. These samples were just pairs of two tubes so it was already a feat to keep everything straight.
• All pooling was done at room temperature since cDNA libraries should be relatively stable.

Once I pooled my samples, I put them back in the -20ºC. I sent them out on dry ice the next day so now we wait for data!

Going forward

1. Examine HOBO data from 2023 experiment
2. Demographic data analysis for 2023 paper
3. Start methods and results of 2023 paper