Laura’s Notebook: QuantSeq Library Generation Batches 3 and 4

IMG_9282

IMG_9283

Batch 3 Sample Information

Sample order in plate Sample No. [RNA] (ng/ul) Vol RNA used Vol H2O to add ng RNA used
1 35 94.4 3.71 1.29 350
2 524 63.0 5.00 315
3 453 196.0 1.79 3.21 350
4 554 188.0 1.86 3.14 350
5 442b 69.8 5.00 349
6 489 68.0 5.00 340
7 462b 106.0 3.30 1.70 350
8 533 6.5 5.00 32.6
9 522 32.2 5.00 161
10 474 77.2 4.53 0.47 350
11 452b 97.2 3.60 1.40 350
12 443 60.2 5.00 301
13 477 164.0 2.13 2.87 350
14 526 138.0 2.54 2.46 350
15 562 126.0 2.78 2.22 350
16 432 74.0 4.73 0.27 350
17 37 74.6 4.69 0.31 350
18 413 130.0 2.69 2.31 350
19 45 25.1 5.00 125.5
20 561 28.0 5.00 140
21 542 32.8 5.00 164
22 527 124.0 2.82 2.18 350
23 492 82.6 4.24 0.76 350
24 475 27.4 5.00 137
25 541 44.4 5.00 222
26 565 31.2 5.00 156
27 B3 – NTC1 NA 5.00 #VALUE!
28 B3 – NTC2 NA 5.00 #VALUE!

Batch 3 Plate Configuration

1 2 3 4 5 6 7 8
A 35 524 453 554 442b 489 462b
B
C 533 522 474 452b 443 477 526
D
E 562 432 37 413 45 561 542
F
G 527 492 475 541 565 B3 – NTC1 B3 – NTC2
H

Batch 4 Sample Information

Sample order in plate Sample No. [RNA] (ng/ul) Vol RNA used Vol H2O to add ng RNA used
1 571 LOW 5.00 NA
2 525 140.0 2.50 2.50 350
3 563 47.0 5.00 235
4 404 112.0 3.13 1.88 350
5 484 58.4 5.00 292
6 531 95.4 3.67 1.33 350
7 34 104.0 3.37 1.63 350
8 490 186.0 1.88 3.12 350
9 523 71.4 4.90 0.10 350
10 473 124.0 2.82 2.18 350
11 485 19.1 5.00 95.5
12 402 114.0 3.07 1.93 350
13 487 118.0 2.97 2.03 350
14 476 39.2 5.00 196
15 421 57.6 5.00 288
16 553 186.0 1.88 3.12 350
17 41 108.0 3.24 1.76 350
18 46 98.6 3.55 1.45 350
19 551 96.4 3.63 1.37 350
20 486b 148.0 2.36 2.64 350
21 445 160.0 2.19 2.81 350
22 43 156.0 2.24 2.76 350
23 506 29.2 5.00 146
24 482 22.2 5.00 111
25 412 31.2 5.00 156
26 488 60.0 5.00 300
27 47 128.0 2.73 2.27 350
28 B4 – NTC2 NA 5.00 0

Batch 4 Plate Configuration

1 2 3 4 5 6 7 8
A 571 525 563 404 484 531 34
B
C 490 523 473 485 402 487 476
D
E 421 553 41 46 551 486b 445
F
G 43 506 482 412 488 47 B4 – NTC2
H

Notes

  • Aliquoted 20 uL RNA into new tubes and froze in -80. Except, for the following samples I aliquoted 7 uL: 34, 35, 41, 46, 46
  • Sample #35 was not fully sealed during either the RNA removal step or the second strand synthesis step, and I believe I lost some volume to evaporation. I proceeded normally, but will see how that library looks.
  • Not sure if I mentioned this previously, but I’m using 350 ng RNA, except for low concentration samples (I use 5 uL to get the maximum amount of RNA possible).
  • To “quickly spin down” samples at room temperature I put the sample plate in the centrifuge, push start, then push stop immediately when the speed does not read 0. I also set the temperature to room temperature so it doesn’t actively cool.

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Laura’s Notebook: QuantSeq Purification and qPCR Assay Batches 1 & 2

ds cDNA purification, pre-PCR

I purified the ds cDNA from batches 1 and 2. Notes on how to improve that process:

  • Bubbles! Bubbles make it difficult to use a mutichannel pipette. Need to improve handling to minimize bubble formation.
  • Should reduce amount of time I keep samples on magnet and to dry. The beads seem to crack a bit, and it’s difficult to resuspend them in the final elution step.
  • Max # of PCR plate columns for purification step = 8

qPCR assay for optimal endpoint PCR cycles

I performed the qPCR assay on all the ctenidia samples (batches 1 and 2). Here are the amplification curves:

image

Batch 1 end-point PCF cycle calculations

Sample No. 50% max No. Cycles @ 50% No. Cycles @ 50% minus 3 cycles Cycles, round down
328 1,662 16.51 13.51 13
299 1,603 18.54 15.54 15
301 1,572 18.66 15.66 15
342 1,673 18.85 15.85 15
331 1,572 18.94 15.94 16
307 1,001 28.77 25.77 25
295 1,498 21.86 18.86 18
qPCR NTC 1,352 29.1 26.1 26
304 1,392 18.05 15.05 15
305 1,334 19.41 16.41 16
311 1,449 19.09 16.09 16
NTC2 – B1 1,521 25.92 22.92 23
298 1,437 18.27 15.27 15
348 1,394 19.33 16.33 16
315 1,496 18.74 15.74 15
qPCR NTC 1,483 28.23 25.23 25
344 1,545 18.19 15.19 15
325 2,029 17.64 14.64 14
338 1,572 19.31 16.31 16
347 1,547 17.9 14.9 15
312 1,510 18.77 15.77 15
321 1,561 18.03 15.03 15
333 1,651 18.07 15.07 15
291 1,265 18.06 15.06 15
308 1,297 19.1 16.1 16
NTC1 – B1 1,538 25.23 22.23 22
335 1,449 18.86 15.86 15
318 1,586 19.01 16.01 16
294 1,444 19.3 16.3 16
324 1,648 19.34 16.34 16

Batch 2 end-point PCR cycle calculations

Sample No. RFU @ endpoint 50% max No. Cycles @ 50% No. Cycles @ 50% minus 3 cycles Cycles, round down
343 3583 1791.5 16.62 13.62 13
345 4129 2,065 17.31 14.31 14
303 3543 1,772 17.28 14.28 14
346 2823 1,412 17.75 14.75 14
302 3153 1,577 16.81 13.81 13
336 3300 1,650 22.82 19.82 19
292 3253 1,627 18.6 15.6 15
NTC1 -B2 3079 1,540 20.67 17.67 17
317 3576 1,788 18.8 15.8 15
322 2540 1,270 19.31 16.31 16
332 2689 1,345 20.02 17.02 17
334 2851 1,426 20.78 17.78 17
349 2825 1,413 18.57 15.57 15
337 3325 1,663 18.65 15.65 15
341 3152 1,576 17.61 14.61 14
313 3184 1,592 18.47 15.47 15
309 2962 1,481 18.05 15.05 14
327 2973 1,487 18.89 15.89 15
319 3037 1,519 20.88 17.88 16
326 2810 1,405 18.12 15.12 15
306 3698 1,849 21.19 18.19 18
323 3537 1,769 18.47 15.47 15
314 2996 1,498 20.58 17.58 17
316 2929 1,465 19.02 16.02 16
339 2761 1,381 19.33 16.33 16
293 2213 1,107 19.67 16.67 16
329 2164 1,082 18.72 15.72 15
296 2182 1,091 18.1 15.1 15

Notes

  • The highest quality RNA samples require 12 cycles. According to my QuantSeq rep, the very lowest quality and concentration RNA samples require 25 cycles.
  • I should use the optimal cycle number for end-point PCR, BUT some people that are in a hurry run all samples using the average of three consecutive cycles. For instance, if some samples need 14, some need 15, and some need 16, one can run all samples for 15 cycles. However, that is not the optimal protocol. I will proceed, but may need to re-generate a few libraries, which I will determine using Qubut and high sensitivity DNA chip on the Bioanalyzer.

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