Equal-Molar Pooling of mRNA Libraries for Sequencing
Libraries from cells and flies infected with DiNV were prepared for sequencing here, here, here, and here
6 samples from each the flies and the cells libraries were checked for the peak distribution on a TapeStation (see here for TS), and so the average library size was calculated for all the libraries. This allowed to calculate the nM concentration of each library when using the size and the qubit value.
I then decided to dilute the libraries to 10nM for pooling, so that each library is at 10nM and is combined together. These were diluted in a total of 100ul (each containing 1000nMoles of library)
First the fly libraries were thawed on ice, then diluted in separate tubes with TE buffer to 10nM on ice . Then, 5ul from each diluted library was combined togethr in one 1.5mL tube :
| sample number | nM conc | ul for 1000nMoles | ul TE to 100ul | ul pooled together |
|---|---|---|---|---|
| 1 | 256.5 | 3.9 | 96.1 | 5 |
| 2 | 210.5 | 4.7 | 95.3 | 5 |
| 6 | 155.9 | 6.4 | 93.6 | 5 |
| 7 | 158.4 | 6.3 | 93.7 | 5 |
| 16 | 179.9 | 5.6 | 94.4 | 5 |
| 17 | 197.8 | 5.1 | 94.9 | 5 |
| 26 | 144.1 | 6.9 | 93.1 | 5 |
| 11 | 119.6 | 8.4 | 91.6 | 5 |
| 12 | 147.2 | 6.8 | 93.2 | 5 |
| 21 | 97.1 | 10.3 | 89.7 | 5 |
| 22 | 109.9 | 9.1 | 90.9 | 5 |
| 31 | 115.0 | 8.7 | 91.3 | 5 |
| 3 | 190.0 | 5.3 | 94.7 | 5 |
| 4 | 269.0 | 3.7 | 96.3 | 5 |
| 8 | 144.5 | 6.9 | 93.1 | 5 |
| 9 | 146.6 | 6.8 | 93.2 | 5 |
| 18 | 116.2 | 8.6 | 91.4 | 5 |
| 19 | 141.4 | 7.1 | 92.9 | 5 |
| 27 | 122.9 | 8.1 | 91.9 | 5 |
| 28 | 122.9 | 8.1 | 91.9 | 5 |
| 29 | 120.8 | 8.3 | 91.7 | 5 |
| 13 | 86.2 | 11.6 | 88.4 | 5 |
| 14 | 86.7 | 11.5 | 88.5 | 5 |
| 23 | 101.2 | 9.9 | 90.1 | 5 |
| 24 | 128.5 | 7.8 | 92.2 | 5 |
| 32 | 92.4 | 10.8 | 89.2 | 5 |
| 33 | 62.5 | 16.0 | 84.0 | 5 |
| 34 | 107.9 | 9.3 | 90.7 | 5 |
| 140 |
Then the cell libraries were thawed on ice, then diluted in separate tubes with TE buffer to 10nM on ice . Then, 5ul from each diluted library was combined togethr in one 1.5mL tube :
| sample number | nM conc | ul for 1000nMoles | ul TE to 100ul | ul pooled together |
|---|---|---|---|---|
| 9 | 346.4 | 2.9 | 97.1 | 5 |
| 25 | 310.5 | 3.2 | 96.8 | 5 |
| 41 | 323.5 | 3.1 | 96.9 | 5 |
| 13 | 244.2 | 4.1 | 95.9 | 5 |
| 29 | 302.0 | 3.3 | 96.7 | 5 |
| 45 | 366.8 | 2.7 | 97.3 | 5 |
| 1 | 267.6 | 3.7 | 96.3 | 5 |
| 2 | 99.1 | 10.1 | 89.9 | 5 |
| 3 | 103.2 | 9.7 | 90.3 | 5 |
| 4 | 125.3 | 8.0 | 92.0 | 5 |
| 10 | 79.1 | 12.6 | 87.4 | 5 |
| 11 | 95.0 | 10.5 | 89.5 | 5 |
| 12 | 133.0 | 7.5 | 92.5 | 5 |
| 26 | 133.5 | 7.5 | 92.5 | 5 |
| 27 | 86.3 | 11.6 | 88.4 | 5 |
| 28 | 236.3 | 4.2 | 95.8 | 5 |
| 42 | 233.7 | 4.3 | 95.7 | 5 |
| 43 | 198.3 | 5.0 | 95.0 | 5 |
| 44 | 202.9 | 4.9 | 95.1 | 5 |
| 14 | 135.1 | 7.4 | 92.6 | 5 |
| 15 | 148.9 | 6.7 | 93.3 | 5 |
| 16 | 187.5 | 5.3 | 94.7 | 5 |
| 30 | 189.0 | 5.3 | 94.7 | 5 |
| 31 | 204.9 | 4.9 | 95.1 | 5 |
| 32 | 253.7 | 3.9 | 96.1 | 5 |
| 46 | 216.2 | 4.6 | 95.4 | 5 |
| 47 | 234.7 | 4.3 | 95.7 | 5 |
| 48 | 298.4 | 3.4 | 96.6 | 5 |
| P4 | 7.3 | NA | NA | 2 |
| 142 |
The two tubes were combined together into one tube, with all of the libraries at the same nM concentration. This was then frozen at -20 before being sent for sequencing.
All information on these libraries can be found here for cells and here for flies