West Coast Green Crab Experiment Part 66

Continuing first round of library preparation

Just chugging along with library prep!

  • Protocol can be found here. Any updates that I think are necessary from my notes are then added to the protocol.
  • All primer and cDNA concentration information can be found in this spreadsheet.

2025-07-07

Notes

  • Samples: 5-123, 5-002, 25-170, 25-116, 5-008, 5-005, 5-061, 25-168
  • Tagging primers: i7-7, i5-1 through i5-8
  • When I was taking samples out of the -80 I LOST A SAMPLE. Literally dropped it as I was trying to pick up another sample. I searched high and low in the RNA room, but sample 5-064 is no where to be found. This sample was a CT from 5ºC sampled at week 6. Honestly, not too stressed about it since I don’t need the genotype and there are 5 other samples from that timepoint. I refuse to re-extract at this point.
  • Added a bit more than 5 µL of the fragmented RNA since samples 1-4 weren’t pipetting well with the multichannel (I must not be rocking well)
  • For the first bead dry, samples were dried for 2-2.5 minutes. The centrifuge really separated the pellets out, and the tops were starting to lighten and crack. The pellets themselves weren’t as dry as desired but I didn’t want to have the beads crack any more.
  • Sample 5 was a little frozen after adding the end prep reagents and mixing
  • Sample 3 was very foamy after mixing all of the PCR reagents
  • Dried all samples for 2.5 minutes, except for sample 6 which I dried for 3 minutes during the second bead drying step
  • Qubit S1 = 55.61, S2 = 25316.48

Results

I have really nice yields today!!

While I was working on library prep, I was chatting with Sara to figure out what to do about my libraries with really low concentrations. Given that I don’t have BioAnalyzer reports for my libraries, Sara suggested I re-prep the libraries from scratch instead of trying to re-amplify them. So far, I need to re-prep the following libraries with concentrations < 5 ng/µL:

  • i7-1: i5-1 through i5-8
  • i7-2: i5-1, i5-2, i5-6
  • i7-3: i5-1 through i5-8
  • i7-5: i5-3, i5-4, i5-5, i5-6, i5-7
  • i7-12: i5-1

I will have i7-11 i5-3 through i5-8 and i7-12 i5-6 through i5-8 leftover once I finish my first round of everything. This means I can redo all of my i7-1 and i7-12_i5-1 with these leftover indices. Today, I also noticed that there’s an extra 5 µL of i7-7 after I finished all my samples. That means that I have ~1 sample worth of all my finished i7 indices. However, I’ll still need my i7-2, i7-3, and i7-5 indices for more than one sample, as well as extra i5-1 through i5-8. Since I need so many indices, I need to buy more tagging primers! I emailed NEB tech support to see if there is a way to get just what indices I need, instead of ordering the full 96 sample kit again.

I will also need more reagents to complete these libraries. I have 87 samples total, but I prepped 6 with reagents from the sample kit. That gives me 81 samples + 15 re-preps = 96 total samples that I would need reagents for. I mayyyy be able to eek out okay with what i have in the kit, but I asked Carolyn to order a sample PolyA module and library prep kit just in case, which will give me reagents for 6 more samples. I also asked Sara if the kit usually has extra reagents for a sample or two.

I did spill Tris and the reconstitution buffer. I should be fine on Tris, but I need to triple check my reconstitution buffer volume to ensure I have enough for the re-preps (or at the very least, to finish out this kit: 5 x 8 = 40 samples).

2025-07-08

Notes

  • Samples: 25-110, 25-119, 5-189, 5-182, 5-063, 5-007, 5-071, 25-174
  • Tagging primers: i7-8, i5-1 through i5-8
  • The printer was being a real pain in my ass so I had to reuse the protocol from yesterday. All of my notes from today are in red pen.
  • Sample 6 had way too much buffer in it than the rest of the samples after I added 50 µL of Tris and Binding buffer? My guess is that there was too much wash buffer leftover in this sample, since the Tris and Binding buffer aliquots all had equal volumes. I tried pipet mixing but I flicked the samples due to the high volume of sample 6.
  • Added a bit more than 6.5 µL of fragmentation master mix due to a 10 µL multichannel pipetting issue
  • There was a fire drill while my samples were in the thermocycler for first strand synthesis! I was delayed by 5 minutes in starting the second strand synthesis since I had to taw the Second Strand Master Mix.
  • Samples were dried for ~2.5 minutes during the first bead dry due to the beads being very separate and the top starting to lighten and crack
  • Checked if I have enough reconstitution buffer for 40 samples (40 x 40 = 160 µL). I pulled out 1000 µL of reconstitution buffer, so I should be fine?
  • Samples 1-5 were dried for 2 minutes and samples 6-8 were dried for 3 minutes. I’ve finally figured out the secret to bead drying! They have to look dark, but more lumpy instead of fully glossy and smooth.
  • Qubit S1 = 56.37, S2 = 24570.12

Results

Great yields today! Interestingly, the weirdness with sample 6 didn’t translate to a reduced yield.

Carolyn is working on getting me extra reagents, and I reached out to NEB technical support to see if I needed to buy new tagging primers or not.

2025-07-09

Notes

  • Samples: 25-060, 25-179, 5-073, 5-126, 25-051, 25-053, 5-197, 25-059
  • Tagging primers: i7-9, i5-1 through i5-8
  • Forgot to wait until temperature reached 65ºC after fragmentation to put tubes on ice! I held them outside the ice for a bit, hopefully it doesn’t mess with too much.
  • During the first bead clean, samples 6-8 didn’t clump well after centrifuging and trying to slide the beads back onto the magnet. I dried beads for 2 minutes. There were faint streaks of beads that didn’t get mixed with the liquid and not on the bottom of the tube in samples 1 and 7 after I eluted.
  • I have ~1100 µL of reconstitution buffer left. This will be enough for 26 samples. I also have the sample kit, which should give me 32 samples of reconstitution buffer. That means I need reconstitution buffer for 11 more samples (~450 µL). I asked Sara to see if Harriet’s lab has any extra. If they do not, I will need to order some. I will contact Tech Support to see if I can purchase just this buffer.
  • NEB Technical Support suggested mixing single index with dual index primers if I was trying to multiplex a small number of samples. Unfortunately, that is not the case so I ordered more tagging primers. Will need to see if Harriet wants what is leftover.
  • Dried samples 7 and 8 for 2 minutes, 3 for 2.5 minutes, 1 and 2 for 3 minutes, 4 and 5 for 3.5 minutes, and 6 for 4 minutes during the second bead drying step.
  • Qubit S1 = 55.58, S2 = 26546.04

Results

Yields look good! Some better than others but I don’t know why.

2025-07-10

Notes

  • Samples: 25-113, 25-048, 25-186, 25-185, 25-172, 5-067, 5-006, 5-135
  • Tagging primers: i7-10, i5-1 through i5-8
  • I have about 500 µL of beads left, which gets me enough for 25 samples. Since I usually add a little extra to the beads, this gives me close to 3 sets of full samples. I’ll see if I can change the math to add samples + 1/2 extra instead of a fulll extra sample to stretch this out.
  • Added a little extra fragmentation mix to sample 8 due to my inability to still use a multichannel pipet.
  • Started the fragmentation program on the thermocycler 30 seconds to 1 minute late because I forgot to actually press start before going to get ice!
  • Spilled some of the reconsituted beads after the first bead drying step behind the tubes on the mag stand. I was able to recover them! I also cleaned the mag stand again before proceeding.
  • Final bead dry was between 2.5 and 3.5 minutes
  • Qubit S1 = 54.49, S2 = 25891.25

Results

AWESOME yields today! For fun, I did the Qubit with the 1x dSDNA HS program and the dsDNA HS program. They gave yields within ~2 µL of eachother, with the 1x program having relatively higher yields. It was a fun experiment but doesn’t mean anything.

I was talking to Sara, and it may be worth trying to re-amplify the samples with yields between 3-5 ng/µL to see if that improves yields since I already bought a new set of indices. Otherwise, I may have to buy a 24 sample kit to finish everything off, which would entail really looking at the budget again and (maybe) trying to find a bit more money somewhere.

I also moved all samples that need to be re-prepared in some way to a box, and mapped out which samples will need to use which indices.

2025-07-11

Today I finished out the last two first round samples and re-prepped the majority of the samples with i7-1 primers since those yields were abysmal. I also took inventory of how much of each reagent I have left so I can determine what I may need to purchase. I have 18 samples left after today that need to be re-prepped!

Notes

  • Samples: 5-188, 5-128, 25-056, 25-194, 25-049, 25-203, 15-152, 25-167
  • Tagging primers: i7-11, i5-1 through i5-8
  • I checked to see if I had enough reagents for 30 samples in this kit itself, since I have 26 samples left to finish
    • RNA Fragmentation Mix: > 80 µL –> enough!
    • dT25 Beads: 400 µL –> ~ 20 samples. I am going to eek this out as much as possible by doing 1 sample + 0.25 extra
    • RNA Binding Buffer: > 18 mL –> enough!
    • Wash Buffer: more than enough
    • Tris Buffer: enough!
    • Strand Specificity Reagent: > 110 µL –> enough!
    • First Strand Synthesis Enzyme Mix: > 30 µL –> enough!
    • Second Strand Master Mix: > 900 µL –> enough!
    • Ampure beads: > 7 mL –> enough!
    • TE Buffer: > 3 mL –> enough!
    • End Prep Reaction Buffer: > 75 µL –> enough!
    • End Prep Enzyme Mix: ~ 30 µL –> this is enough for 20 samples. With the sample kit, I should be able to squeeze by just enough
    • Adaptor, Dilution Buffer, and USER enzyme: I have an obscene amount
    • Ligation Master Mix: > 360 µL –> enough!
    • MSTC High Yield Master Mix: > 1500 –> enough!
    • Reconstitution Buffer: 425 µL –> this is enough for 10 samples, and I have 6 samples extra. Sara has some more reconstitution buffer, so hopefully I will have enough.
  • Beads were dried for 2-2.5 minutes during both bead drying steps
  • Qubit S1 = 54.40, S2 = 25797.00

Results

Yields for these redone samples look great!

It seems like the things that are limiting me are:

  • Reconstitution buffer: this is what I have the LEAST of. I emailed NEB to see if I can find a way to purchase just the buffer.
  • dT25 Beads and End Prep Enzyme Mix: I may have juuuuuust enough.

Going forward

  1. Re-prep specific libraries with concentration < 5 ng/µL
  2. Pool RNA libraries
  3. Send RNA for sequencing!
  4. Examine HOBO data from 2023 experiment
  5. Demographic data analysis for 2023 paper
  6. Start methods and results of 2023 paper
Written on July 7, 2025