Today I pooled samples for the 6 new libraries for sequencing. There is over 1000ng in each pooled sample, so everything is ready to go! Details in post.
Pooling
I pooled 110ng of RNA from 10 samples within each of the 6 libraries. Math below:
| FRP | pool_number | trtmnt_tank | day | infection_status | maturity | tube_number | total-yield_ng | elution_vol | vol_for_pool |
|---|---|---|---|---|---|---|---|---|---|
| 6112 | 5 | NA | 9 | 0 | I | 28 | 287.3 | 13 | 4.977 |
| 6136 | 5 | NA | 9 | 0 | M | 91 | 204.1 | 13 | 7.006 |
| 6143 | 5 | NA | 9 | 0 | M | 10 | 297.7 | 13 | 4.803 |
| 6150 | 5 | NA | 9 | 0 | M | 119 | 419.9 | 13 | 3.406 |
| 6161 | 5 | NA | 9 | 0 | M | 53 | 405.6 | 13 | 3.526 |
| 6213 | 5 | NA | 9 | 0 | I | 108 | 176.8 | 13 | 8.088 |
| 6106 | 5 | NA | 9 | 0 | M | 5 | 174.2 | 13 | 8.209 |
| 6156 | 5 | NA | 9 | 0 | I | 24 | 366.6 | 13 | 3.901 |
| 6157 | 5 | NA | 9 | 0 | M | 169 | 365.3 | 13 | 3.915 |
| 6178 | 5 | NA | 9 | 0 | M | 124 | 296.4 | 13 | 4.825 |
| 6122 | 6 | NA | 9 | 1 | I | 133 | 305.5 | 13 | 4.681 |
| 6201 | 6 | NA | 9 | 1 | I | 163 | 211.9 | 13 | 6.748 |
| 6206 | 6 | NA | 9 | 1 | I | 103 | 208 | 13 | 6.875 |
| 6174 | 6 | NA | 9 | 1 | I | 20 | 360.1 | 13 | 3.971 |
| 6177 | 6 | NA | 9 | 1 | I | 54 | 327.6 | 13 | 4.365 |
| 6187 | 6 | NA | 9 | 1 | I | 158 | 241.8 | 13 | 5.914 |
| 6199 | 6 | NA | 9 | 1 | I | 151 | 241.8 | 13 | 5.914 |
| 6231 | 6 | NA | 9 | 1 | I | 102 | 188.5 | 13 | 7.586 |
| 6233 | 6 | NA | 9 | 1 | I | 117 | 300.3 | 13 | 4.762 |
| 6237 | 6 | NA | 9 | 1 | I | 17 | 226.2 | 13 | 6.322 |
| 6104 | 7 | cold | 12 | 0 | I | 259 | 412.1 | 13 | 3.470 |
| 6106 | 7 | cold | 12 | 0 | M | 241 | 169 | 13 | 8.462 |
| 6153 | 7 | cold | 12 | 0 | M | 209 | 315.9 | 13 | 4.527 |
| 6157 | 7 | cold | 12 | 0 | M | 224 | 249.6 | 13 | 5.729 |
| 6160 | 7 | cold | 12 | 0 | M | 238 | 494 | 13 | 2.895 |
| 6172 | 7 | cold | 12 | 0 | M | 316 | 373.1 | 13 | 3.833 |
| 6175 | 7 | cold | 12 | 0 | I | 246 | 269.1 | 13 | 5.314 |
| 6178 | 7 | cold | 12 | 0 | M | 218 | 162.5 | 13 | 8.800 |
| 6189 | 7 | cold | 12 | 0 | I | 216 | 577.2 | 13 | 2.477 |
| 6191 | 7 | cold | 12 | 0 | I | 227 | 234 | 13 | 6.111 |
| 6118 | 8 | cold | 12 | 1 | I | 240 | 280.8 | 13 | 5.093 |
| 6128 | 8 | cold | 12 | 1 | I | 231 | 492.7 | 13 | 2.902 |
| 6141 | 8 | cold | 12 | 1 | I | 249 | 241.8 | 13 | 5.914 |
| 6148 | 8 | cold | 12 | 1 | I | 213 | 442 | 13 | 3.235 |
| 6149 | 8 | cold | 12 | 1 | I | 226 | 340.6 | 13 | 4.198 |
| 6163 | 8 | cold | 12 | 1 | I | 228 | 364 | 13 | 3.929 |
| 6164 | 8 | cold | 12 | 1 | I | 257 | 353.6 | 13 | 4.044 |
| 6173 | 8 | cold | 12 | 1 | I | 210 | 432.9 | 13 | 3.303 |
| 6174 | 8 | cold | 12 | 1 | I | 233 | 457.6 | 13 | 3.125 |
| 6177 | 8 | cold | 12 | 1 | I | 250 | 410.8 | 13 | 3.481 |
| 6223 | 9 | warm | 12 | 0 | M | 270 | 380.9 | 13 | 3.754 |
| 6228 | 9 | warm | 12 | 0 | I | 290 | 370.5 | 13 | 3.860 |
| 6230 | 9 | warm | 12 | 0 | M | 292 | 223.6 | 13 | 6.395 |
| 6232 | 9 | warm | 12 | 0 | M | 286 | 663 | 13 | 2.157 |
| 6234 | 9 | warm | 12 | 0 | I | 263 | 345.8 | 13 | 4.135 |
| 6235 | 9 | warm | 12 | 0 | I | 297 | 531.7 | 13 | 2.689 |
| 6238 | 9 | warm | 12 | 0 | M | 375 | 301.6 | 13 | 4.741 |
| 6242 | 9 | warm | 12 | 0 | M | 377 | 289.9 | 13 | 4.933 |
| 6253 | 9 | warm | 12 | 0 | I | 275 | 387.4 | 13 | 3.691 |
| 6265 | 9 | warm | 12 | 0 | I | 282 | 317.2 | 13 | 4.508 |
| 6231 | 10 | warm | 12 | 1 | I | 278 | 230.1 | 13 | 6.215 |
| 6233 | 10 | warm | 12 | 1 | I | 371 | 370.5 | 13 | 3.860 |
| 6244 | 10 | warm | 12 | 1 | I | 284 | 358.8 | 13 | 3.986 |
| 6245 | 10 | warm | 12 | 1 | I | 365 | 481 | 13 | 2.973 |
| 6249 | 10 | warm | 12 | 1 | I | 279 | 461.5 | 13 | 3.099 |
| 6255 | 10 | warm | 12 | 1 | I | 262 | 536.9 | 13 | 2.663 |
| 6256 | 10 | warm | 12 | 1 | I | 283 | 247 | 13 | 5.789 |
| 6257 | 10 | warm | 12 | 1 | I | 363 | 269.1 | 13 | 5.314 |
| 6261 | 10 | warm | 12 | 1 | I | 273 | 469.3 | 13 | 3.047 |
| 6262 | 10 | warm | 12 | 1 | I | 289 | 352.3 | 13 | 4.059 |
Results:
Ran 2ul of each pooled sample on Qubit RNA Hs
| trtmnt_tank | day | infection_status | total-yield_ng | elution_vol | vol_for_pool | total_vol_ul | qubit_vol_ul | qubit_sample_ng | total_ng_RNA_pool | final_sample_vol |
|---|---|---|---|---|---|---|---|---|---|---|
| NA | 9 | 0 | 287.3 | 13 | 4.977375566 | 52.65550411 | 2 | 21 | 1063.765586 | 50.65550411 |
| NA | 9 | 1 | 305.5 | 13 | 4.680851064 | 57.13842355 | 2 | 21.5 | 1185.476106 | 55.13842355 |
| cold | 12 | 0 | 412.1 | 13 | 3.470031546 | 51.61757335 | 2 | 23.6 | 1170.974731 | 49.61757335 |
| cold | 12 | 1 | 280.8 | 13 | 5.092592593 | 39.22471819 | 2 | 27.2 | 1012.512335 | 37.22471819 |
| warm | 12 | 0 | 380.9 | 13 | 3.754266212 | 40.86453844 | 2 | 26.9 | 1045.456084 | 38.86453844 |
| warm | 12 | 1 | 230.1 | 13 | 6.214689266 | 41.00446376 | 2 | 26.3 | 1025.817397 | 39.00446376 |
There’s at least 1000ng of RNA in all pooled samples!! :fireworks: :tada: