Grace’s Notebook: January 2020 Goals

Crab

  • Concentrate the 6 new pooled crab samples (recieved that sample kit of the RNA Clean and Concentrator -5 kit from Zymo – have a lot of time tomorrow to work on this)
  • Submit 6 new pooled samples (to NWGC because we have the quote from them)
  • Work on new results for AMSS presentation (my talk needs to be done by Monday, January 27th)

Oyster

  • FINISH PAPER –> just do it!

Other

  • Submit official MS thesis proposal (nearly completed)
  • Submit application for SAFS fellowship (have list of things to submit. nearly complete – just waiting on me finishing the MS thesis proposal and a few other easy tasks)
  • Figure out how to balance TA commitments with my thesis work and class work (taking Global Health 518- Understanding and Managing the Health Risks of Climate Change (human health))

Attend and present at AMSS 2020 January 27th – 30th

  • All my paperwork is in order. Waiting to register for conference until week before (Pam’s suggestion), and my talk slot is Tuesday, January 28th 9:15-9:30am.

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

from The Shell Game https://ift.tt/2ZXDcp2
via IFTTT

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

from The Shell Game https://ift.tt/2SUc217
via IFTTT