Laura’s Notebook: QuantSeq – inventory of finished libraries

Library prep is complete! However, I will need to redo many 😦 Based on Bioanalyzer results, any libraries with concentration <1.0 ng/uL will need to be re-done. Out of the total 132 samples that I prepped, 34 had concentrations that were LOW or <1.0 ng/uL (according to Qubit). That’s about a 25% incompletion rate. Here is the final inventory.

Next step – redo the 34 samples. Need to decide whether I should re-isolate RNA, or just try to re-do the library generation. This is a question for lab meeting tomorrow.

Ctenidia

Cohort pCO2 HOMOGENATE TUBE # TISSUE TYPE TISSUE SAMPLE # [RNA] after Dnase treatment (ng/uL) End Point PCR cycles ds cDNA concentration
Dabob Bay High 291 CTENIDIA HL10-10 158.0 15 LOW
Dabob Bay High 292 CTENIDIA HL10-11 29.6 15 1.32
Dabob Bay High 293 CTENIDIA HL10-12 39.6 16 1.03
Dabob Bay High 294 CTENIDIA HL6-10 110.0 16 1.95
Dabob Bay High 295 CTENIDIA HL6-11 34.8 18 LOW
Dabob Bay High 296 CTENIDIA HL6-12 180.0 15 3.46
Dabob Bay High 298 CTENIDIA HL6-14 182.0 15 3.08
Dabob Bay High 299 CTENIDIA HL6-15 50.4 15 2.46
Dabob Bay Ambient 301 CTENIDIA HL10-19 75.8 15 2.82
Dabob Bay Ambient 302 CTENIDIA HL10-20 62.4 14 4.90
Dabob Bay Ambient 306 CTENIDIA HL10-21 136.0 18 LOW
Dabob Bay Ambient 304 CTENIDIA HL6-19 200.0 15 2.80
Dabob Bay Ambient 305 CTENIDIA HL6-20 75.2 16 2.96
Dabob Bay Ambient 303 CTENIDIA HL6-21 95.2 14 2.40
Dabob Bay Ambient 307 CTENIDIA HL6-16 89.4 TOO LOW TOO LOW
Dabob Bay Ambient 308 CTENIDIA HL6-17 73.6 16 2.48
Dabob Bay Ambient 309 CTENIDIA HL6-18 170.0 14 1.46
Oyster Bay High 311 CTENIDIA SN6-16 158.0 16 3.76
Oyster Bay High 312 CTENIDIA SN6-17 90.6 15 1.58
Oyster Bay High 313 CTENIDIA SN6-18 72.4 15 1.81
Oyster Bay High 314 CTENIDIA SN6-19 42.2 17 LOW
Oyster Bay High 315 CTENIDIA SN6-20 148.0 15 2.32
Oyster Bay High 316 CTENIDIA SN6-21 146.0 16 3.74
Oyster Bay High 317 CTENIDIA SN6-22 158.0 15 2.14
Oyster Bay High 318 CTENIDIA SN6-23 174.0 16 4.08
Oyster Bay High 319 CTENIDIA SN6-24 77.6 16 0.64
Oyster Bay Ambient 321 CTENIDIA SN6-25 148.0 15 3.64
Oyster Bay Ambient 322 CTENIDIA SN6-26 44.6 16 NOT QUANTIFIED
Oyster Bay Ambient 323 CTENIDIA SN6-27 102.0 15 3.70
Oyster Bay Ambient 324 CTENIDIA SN6-28 172.0 16 0.78
Oyster Bay Ambient 325 CTENIDIA SN6-29 180.0 14 2.92
Oyster Bay Ambient 326 CTENIDIA SN6-30 130.0 15 4.04
Oyster Bay Ambient 327 CTENIDIA SN6-31 85.2 15 1.97
Oyster Bay Ambient 328 CTENIDIA SN6-32 156.0 14 2.12
Oyster Bay Ambient 329 CTENIDIA SN6-33 162.0 15 0.33
Fidalgo Bay High 331 CTENIDIA NF6-16 42.2 16 LOW
Fidalgo Bay High 332 CTENIDIA NF6-17 65.8 17 LOW
Fidalgo Bay High 333 CTENIDIA NF6-18 78.6 15 2.98
Fidalgo Bay High 334 CTENIDIA NF6-19 64.8 17 LOW
Fidalgo Bay High 335 CTENIDIA NF6-20 180.0 15 2.58
Fidalgo Bay High 336 CTENIDIA NF6-21 94.8 20 LOW
Fidalgo Bay High 337 CTENIDIA NF6-22 194.0 15 4.02
Fidalgo Bay High 338 CTENIDIA NF6-23 81.6 16 2.74
Fidalgo Bay High 339 CTENIDIA NF6-24 77.2 16 1.91
Fidalgo Bay Ambient 341 CTENIDIA NF6-25 89.6 14 1.31
Fidalgo Bay Ambient 342 CTENIDIA NF6-26 162.0 15 1.78
Fidalgo Bay Ambient 343 CTENIDIA NF6-27 114.0 14 2.58
Fidalgo Bay Ambient 344 CTENIDIA NF6-28 25.0 15 1.58
Fidalgo Bay Ambient 345 CTENIDIA NF6-29 190.0 14 3.50
Fidalgo Bay Ambient 346 CTENIDIA NF6-30 43.6 14 1.36
Fidalgo Bay Ambient 347 CTENIDIA NF6-31 69.0 15 LOW
Fidalgo Bay Ambient 348 CTENIDIA NF6-32 54.4 16 3.06
Fidalgo Bay Ambient 349 CTENIDIA NF6-33 82.0 15 1.90

Larvae

Spawning Tank Cohort Treatment Homo./RNA TUBE # [RNA] after Turbo Dnase treatment (ng/uL) End Point PCR cycles ds cDNA concentration
HL-10 Ambient Dabob Bay 10 Ambient 401 93.4 16 1.24
HL-10 Ambient Dabob Bay 10 Ambient 402 114.0 16 2.76
HL-10 Ambient Dabob Bay 10 Ambient 403 136.0 14 4.16
HL-10 Ambient Dabob Bay 10 Ambient 404 112.0 15 2.92
HL-10 Low Dabob Bay 10 Low 411 72.6 16 3.08
HL-10 Low Dabob Bay 10 Low 412 31.2 17 LOW
HL-10 Low Dabob Bay 10 Low 413 130.0 15 1.97
HL-10 Low Dabob Bay 10 Low 414 168.0 15 0.86
HL-6 Ambient Dabob Bay 6 Ambient 421 57.6 14 1.63
HL-6 Low Dabob Bay 6 Low 431b 83.0 16 3.36
HL-6 Low Dabob Bay 6 Low 432 74.0 17 LOW
HL-6 Low Dabob Bay 6 Low 434 63.0 15 5.58
NF-10 Ambient A Fidalgo Bay 10 Ambient 441 16.2 15 LOW
NF-10 Ambient A Fidalgo Bay 10 Ambient 442b 69.8 17 LOW
NF-10 Ambient A Fidalgo Bay 10 Ambient 443 60.2 15 1.36
NF-10 Ambient B Fidalgo Bay 10 Ambient 444 70.6 14 4.08
NF-10 Ambient B Fidalgo Bay 10 Ambient 445 160.0 15 4.00
NF-10 Low B Fidalgo Bay 10 Low 451 68.4 14 2.24
NF-10 Low A Fidalgo Bay 10 Low 452b 97.2 16 2.68
NF-10 Low B Fidalgo Bay 10 Low 453 196.0 14 0.97
NF-6 Ambient B Fidalgo Bay 6 Ambient 461b 84.0 14 2.02
NF-6 Ambient A Fidalgo Bay 6 Ambient 462b 106.0 16 0.21
NF-6 Low B Fidalgo Bay 6 Low 471b 108.0 15 5.20
NF-6 Low B Fidalgo Bay 6 Low 472b 97.0 15 2.42
NF-6 Low B Fidalgo Bay 6 Low 473 124.0 14 2.34
NF-6 Low A Fidalgo Bay 6 Low 474 77.2 14 3.02
NF-6 Low A Fidalgo Bay 6 Low 475 27.4 14 2.44
NF-6 Low A Fidalgo Bay 6 Low 476 39.2 14 4.24
NF-6 Low A Fidalgo Bay 6 Low 477 164.0 15 2.28
SN-10 Ambient A Oyster Bay C1 10 Ambient 481 89.2 14 0.88
SN-10 Ambient B Oyster Bay C1 10 Ambient 482 22.2 14 1.42
SN-10 Ambient B Oyster Bay C1 10 Ambient 483 99.0 16 1.22
SN-10 Ambient A Oyster Bay C1 10 Ambient 484 58.4 15 1.67
SN-10 Ambient B Oyster Bay C1 10 Ambient 485 19.1 14 2.90
SN-10 Ambient A Oyster Bay C1 10 Ambient 486b 148.0 16 LOW
SN-10 Ambient B Oyster Bay C1 10 Ambient 487 118.0 16 1.27
SN-10 Ambient A Oyster Bay C1 10 Ambient 488 60.0 14 3.06
SN-10 Ambient A Oyster Bay C1 10 Ambient 489 68.0 15 3.84
SN-10 Ambient B Oyster Bay C1 10 Ambient 490 186.0 14 2.32
SN-10 Ambient B Oyster Bay C1 10 Ambient 491 58.4 15 6.64
SN-10 Ambient B Oyster Bay C1 10 Ambient 492 82.6 15 2.02
SN-10 Low B Oyster Bay C1 10 Low 41 108.0 17 LOW
SN-10 Low B Oyster Bay C1 10 Low 43 156.0 15 2.48
SN-10 Low B Oyster Bay C1 10 Low 46 98.6 18 LOW
SN-10 Low B Oyster Bay C1 10 Low 47 128.0 17 LOW
SN-10 Low A Oyster Bay C1 10 Low 44 41.4 TOO LOW TOO LOW
SN-10 Low B Oyster Bay C1 10 Low 506 29.2 15 1.63
SN-10 Low A Oyster Bay C1 10 Low 45 25.1 20 LOW
SN-6 Ambient B Oyster Bay C1 6 Ambient 35 94.4 20 LOW
SN-6 Ambient B Oyster Bay C1 6 Ambient 513 142.0 15 5.92
SN-6 Ambient B Oyster Bay C1 6 Ambient 39 89.8 15 2.04
SN-6 Ambient B Oyster Bay C1 6 Ambient 37 74.6 20 LOW
SN-6 Ambient A Oyster Bay C1 6 Ambient 34 104.0 18 LOW
SN-6 Low A Oyster Bay C1 6 Low 521 66.6 17 LOW
SN-6 Low A Oyster Bay C1 6 Low 522 32.2 16 2.42
SN-6 Low B Oyster Bay C1 6 Low 523 71.4 16 2.16
SN-6 Low B Oyster Bay C1 6 Low 524 63.0 14 3.24
SN-6 Low B Oyster Bay C1 6 Low 525 140.0 14 2.44
SN-6 Low A Oyster Bay C1 6 Low 526 138.0 14 2.20
SN-6 Low B Oyster Bay C1 6 Low 527 124.0 15 1.42
SN-6 Low B Oyster Bay C1 6 Low 528 87.6 14 4.48
SN-6 low A Oyster Bay C1 6 Low 529 56.8 15 2.86
K-10 Ambient Oyster Bay C2 10 Ambient 531 95.4 15 2.10
K-10 Ambient Oyster Bay C2 10 Ambient 532 93.6 14 2.12
K-10 Ambient Oyster Bay C2 10 Ambient 533 6.5 17 LOW
K-10 Low Oyster Bay C2 10 Low 541 44.4 15 1.40
K-10 Low Oyster Bay C2 10 Low 542 32.8 16 2.22
K-10 Low Oyster Bay C2 10 Low 543 162.0 14 5.24
K-6 Ambient Oyster Bay C2 6 Ambient 551 96.4 14 1.69
K-6 Ambient Oyster Bay C2 6 Ambient 552b 74.6 15 1.84
K-6 Ambient Oyster Bay C2 6 Ambient 553 186.0 14 3.28
K-6 Ambient Oyster Bay C2 6 Ambient 554 188.0 14 2.38
K-6 Low Oyster Bay C2 6 Low 561 28.0 17 LOW
K-6 Low Oyster Bay C2 6 Low 562 126.0 17 LOW
K-6 Low Oyster Bay C2 6 Low 563 47.0 15 0.19
K-6 Low Oyster Bay C2 6 Low 564 152.0 14 2.24
K-6 Low Oyster Bay C2 6 Low 565 31.2 17 LOW
homog. Control RNA Control 571 LOW 18 LOW
homog. Control RNA Control 572 TOO LOW TOO LOW

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Laura’s Notebook: QuantSeq library amplification, purification, and QA

The final step in the QuantSeq library prep is to amplify my cDNA libraries (using the optimal cycle number) and then purify. Finally, to assess quantity and quality of finished libraries I use the Qubit High Sensitivity DNA kit to measure cDNA concentration, and the Bioanalyzer High Sensitivity DNA chip kit to measure fragment lengths. I worked in batches based on the number of cycles needed to amplify.

IMPORTANT NOTE: I have 2 sets of indexes with the same numbers. I therefore prepped the ctenidia samples with one set, and the larvae with another set. Therefore, I SHOULD NOT run ctenidia and larval samples in the same lane (since two samples have the same index number).

The spreadsheet where I have all this information organized is in the laura-quantseq repo, file is: 2019-July_RNA-isolation-ctenidia-larvae.xlsx

12/30/2019 – 16 cycles

  • I noticed that I didn’t have the full 17 uL in any of the ds cDNA libraries, probably missing ~ 2uL. Not sure why, possibly due to pipette loss?
  • Sample #331 volume depleted after PCR, maybe due to evaporation b/c there was a small crease in the foil? This was located at well A1 – should ensure tight seal for all future plates.

Amplification plate configuration

Snip20200106_8

1/1/2020 – 14 cycles

  • Again, samples <17 uL.

Amplification plate configuration

Snip20200106_9

1/2/2020 – 15 cycles

  • Again, most samples <17 uL.

Amplification plate configuration

Snip20200106_10

1/4/2020 – 17, 18 and 20 cycles

  • I amplified 3 batches of samples today – those needing 17, 18 and 20 cycles (ran in that order). I held amplified samples at 10C until all groups were done, then combined all samples onto one plate, purified, and quantified.
  • Unfortunately, none of these libraries amplified! I don’t know whether there was an error in the amplification step, OR if the high number of cycle needed represent low concentration/quality. Looking/thinking back through all my steps I do not see any possible errors that could have been made in the PCR mix (it’ a simple master mix using 2 reagents), or during purification. Regardless, I will need to re-do all these libraries!

Amplification plate configuration & index

Snip20200106_11

Snip20200106_12

Snip20200106_13

Amplified and Indexed QuantSeq cDNA libraries

This is the plate configuration of the finished libraries. Based on the quantification/Bioanalyzer results, some of these libraries will need to be re-done. However, this is an important map!

Snip20200106_14

Snip20200106_15

Bioanalyzer quality check

  • I ran 1-2 chips per amplification group. The first 2 chips, which I did for the 16 cycle group, worked great! Results below.
  • I had some issues with the next few chips. Not sure why, but I suspect that it had to do with the fact that I used the Seeb lab’s pipette/pipette tips.
  • I have other chip results, but did not take screen shots at the time – will follow up with those, but for the time being all files are saved in the repo: laura-quantseq/data/library-prep

Results for samples ran for 16 cycles

imageimageSnip20200106_6image

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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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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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Laura’s Notebook: QuantSeq Library Generation Batch 1

Began my full QuantSeq library prep today. I am processing ctenidia samples first, and since there are 53 samples I’m doing ~half at a time. Today I generated double stranded cDNA for 26 samples + 2 NTC (28 total). I loaded samples onto a PCR plate in 4 rows of 7.

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

cDNA synthesis 12/5/19 # samples + 2 NTC 28 Work in 4 rows of 7
Sample order in plate Sample No. [RNA] (ng/ul) Vol RNA used Vol H2O to add ng RNA used
1 328 156.0 2.24 2.76 350
2 299 50.4 5.00 252
3 301 75.8 4.62 0.38 350
4 342 162.0 2.16 2.84 350
5 331 42.2 5.00 211
6 307 89.4 3.91 1.09 350
7 295 34.8 5.00 174
8 304 200.0 1.75 3.25 350
9 305 75.2 4.65 0.35 350
10 311 158.0 2.22 2.78 350
11 NTC2 – B1 5.00 0
12 298 182.0 1.92 3.08 350
13 348 54.4 5.00 272
14 315 148.0 2.36 2.64 350
15 344 25.0 5.00 125
16 325 180.0 1.94 3.06 350
17 338 81.6 4.29 0.71 350
18 347 69.0 5.00 345
19 312 90.6 3.86 1.14 350
20 321 148.0 2.36 2.64 350
21 333 78.6 4.45 0.55 350
22 291 158.0 2.22 2.78 350
23 308 73.6 4.76 0.24 350
24 NTC1 – B1 5.00 0
25 335 180.0 1.94 3.06 350
26 318 174.0 2.01 2.99 350
27 294 110.0 3.18 1.82 350
28 324 172.0 2.03 2.97 350

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 + 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 5 161 23 5
Step 7: SS1 10 322 46 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 9 10 11 12
A 328 299 301 342 331 307 295
B
C 304 305 311 NTC2 – B1 298 348 315
D
E 344 325 338 347 312 321 333
F
G 291 308 NTC1 – B1 335 318 294 324
H

Notes

  • Samples were thawed on wet ice, and vortexed once before use.
  • I loaded the first 3 samples, 328, 299 and 301, onto the PCR plate but then had to wait a few minutes (~3-5) for the rest to thaw.
  • Step #2: held samples at 42C for 14 minutes while preparing master mix. Probably a bit too long.
  • I used a whole PCR Plate, which took up the entire thermocycler space. Next time, I should use partial PCR plates to leave room for a PCR strip to pre-warm the master mix needed for step 3 & 4.
  • The centrifuge gradually cooled as I was using it, which was weird since I didn’t change the temp. I think I need to set temperature to 22C every time I use it, b/c it may default to 4C.
  • I aliquoted 20uL of each sample to new tubes and placed in -80 freezer.

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Laura’s Notebook: Oly methylation analysis, Nov 20, 2019

Revisiting Oly methylation data. We now have two lists of loci:

  • 1) DMLs between two Olympia oyster populations, Hood Canal and South Sound, which were identified using MethylKit.
  • 2) Loci where methylation status is associated with oyster shell length, filtered by a) loci have 10x coverage in all samples, and b) loci have 10x coverage in any sample.

Today I re-plotted heatmaps using MACAU loci, based on feedback from Steven & Katherine:

  • Only use loci with 10x coverage
  • Add heatmaps where samples are NOT ordered by cluster analysis, but instead by 1) tree from MethylKit, and 2) shell length. See my notebook, 06-analyzing-MACAU-results-rev1.html, and here’s one of the new heatmaps, with samples (columns) ordered by shell length, and a barplot of shell length below (red = Hood Canal oysters, green = South Sound oysters).

69304438-6e9ddb00-0bd5-11ea-950a-64b24e80fbf4.png69304447-78bfd980-0bd5-11ea-995a-149f7b6a6b83.png

Then I used bedtools to see where DMLs and MACAU loci are located, see my notebook here:
07-DML-MACAU-annotation.ipynb.

Finally, I began annotating loci locations for DMLs and MACAU loci; see my notebook: 08-Annotations.html. Here’s a barplot showing which features overlap with the DMLs: image

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