Australia Mytilus EecSeq Probe Synthesis Day 1
Planning, Pooling, and Concentrating RNA for EecSeq Probe Synthesis
All steps in are done with Amy Zyck
First step: Aliquoit out 1ug of RNA each from 4 samples from each location and pool together in a locality pool of 4ug
Doing all locations from the samples sent from Australia as well as a pool for the live Mytilus we collected and extracted here. We want to have a starting volume of 25μl for the KAPA Stranded mRNA Seq Kit, so almost all of the pools had to be vacufuged down to either 25μl or less, and then RNase-free water was added to 25μl. These were then re-frozen at -80 because we would not start the library prep until the next day.
| Locality Pool | Sample | Volume (μl) RNA 1μg | Volume of Pool (μl) | Volume Post-Vacufuge (μl) | Volume (μl) Water to 25μl |
|---|---|---|---|---|---|
| Atlantic (AL) | A4 | 7.25 | 38.47 | 25 | 0 |
| - | A5 | 6.33 | - | - | - |
| - | A7 | 10.63 | - | - | - |
| - | 9 | 14.27 | - | - | - |
| France (FR) | 4385 | 29.15 | 157.59 | 20.8 | 4.2 |
| - | 4388 | 32.57 | - | - | - |
| - | 4395 | 39.68 | - | - | - |
| - | 4405 | 56.18 | - | - | - |
| Montenegro (MN) | 4425 | 21.65 | 136.18 | 20 | 5 |
| - | 4415 | 16.67 | - | - | - |
| - | 4445 | 54.95 | - | - | - |
| - | 4452 | 42.92 | - | - | - |
| Tasmania (TZ) | 4598 | 10.29 | 35.89 | 25 | 0 |
| - | 4599 | 11.36 | - | - | - |
| - | 4593 | 3.61 | - | - | - |
| - | 4612 | 10 | - | - | - |
| Rhode Island (RI) | 9R | 5.62 | 20.25 | - | 4.75 |
| - | 10R | 6.76 | - | - | - |
| - | 11R | 4.48 | - | - | - |
| - | 12R | 3.75 | - | - | - |
Second step: Make working stock dilution of adapters
Because we will be using 4000ng of RNA for each library, we needed to make a adapter stock concentration of 1400nM see kit protocol for details.
Our annealed adapters are at 40uM, and we want to make a working stock of 60ul, so the calculation is:
1,400/40,000 * 60 = 2.1μl of adapter stock and 57.9μl of water.
Created working stocks of SAII and NCO adapters.
Third step: Make the plan for adapters and indexes for each locality pool
| Locality Pool | Adapter | Index 1 | Index 2 |
|---|---|---|---|
| AL | SAII | 501 | 701 |
| FR | NCO | 502 | 702 |
| MN | NCO | 503 | 703 |
| TZ | SAII | 504 | 704 |
| RI | NCO | 505 | 705 |