Laura’s Notebook: QuantSeq Library Generation Batch 2

Generated libraries on my second batch of ctenidia RNA samples.

Samples processed, volumes used for RNA and DEPC-treated water, and total RNA used.

Sample order in plate Sample No. [RNA] (ng/ul) Vol RNA used Vol H2O to add ng RNA used
1 343 114.0 3.07 1.93 350
2 345 190.0 1.84 3.16 350
3 303 95.2 3.68 1.32 350
4 346 43.6 5.00 218
5 302 62.4 5.00 312
6 336 94.8 3.69 1.31 350
7 292 29.6 5.00 148
8 NTC1 -B2 5.00 0
9 317 158.0 2.22 2.78 350
10 322 44.6 5.00 223
11 332 65.8 5.00 329
12 334 64.8 5.00 324
13 349 82.0 4.27 0.73 350
14 337 194.0 1.80 3.20 350
15 341 89.6 3.91 1.09 350
16 313 72.4 4.83 0.17 350
17 309 170.0 2.06 2.94 350
18 327 85.2 4.11 0.89 350
19 319 77.6 4.51 0.49 350
20 326 130.0 2.69 2.31 350
21 306 136.0 2.57 2.43 350
22 323 102.0 3.43 1.57 350
23 314 42.2 5.00 211
24 316 146.0 2.40 2.60 350
25 339 77.2 4.53 0.47 350
26 293 39.6 5.00 198
27 329 162.0 2.16 2.84 350
28 296 180.0 1.94 3.06 350

Volumes of solutions needed – I aliquoted volumes into 7 pcr tubes, so I could then add to samples using a multichannel pipette.

Step, Chem. Vol per rxn (uL) Total + 10% or 15% Vol per aliquot (n=7) Vol per sample
Step 3 MM: FS2 9.5 305.9 46 10
Step 3 MM: E1 0.5 16.1
Step 6: RS 5 154 22 5
Step 7: SS1 10 308 44 10
Step 9 MM: SS2 4 128.8 23 5
Step 9 MM: E2 1 32.2

PCR Plate setup

1 2 3 4 5 6 7 8
A 343 345 303 346 302 336 292
B
C NTC1 -B2 317 322 332 334 349 337
D
E 341 313 309 327 319 326 306
F
G 323 314 316 339 293 329 296
H

Notes

  • I worked in 4 rows of 7 (27 samples + 1 NTC).
  • I accidentally used 4.51 uL of sample #326, which is ~585 ug of RNA (exceeds the 500 ug max). I proceeded anyway, and will see if it influences the library quality.
  • Protocol was slightly improved by shortening the amount of time samples sit at 42C at step 2/3 (from 15 mins to ~7 minutes), and I cut the PCR plate to only have 8 of the 12 columns, which freed up space for pre-warming master mix #1 (steps 2/3).
  • I aliquoted 20uL of each sample to new tubes and placed in -80 freezer.

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