Grace’s Notebook: November 2019 Goals

Crab

This coming week:

  • Finish extracting RNA for pools by next week
  • Select samples for 6 pools
    1. Day 9 infected
    2. Day 9 uninfected
    3. Day 12, cold, infected
    4. Day 12, cold, uninfected
    5. Day 12, warm, infected
    6. Day 12, warm, uninfected
  • Run those samples for pooling on bioanalyzer; if they look good, pool samples
  • Have 6 pooled samples ready to send over to NWGC by end of next week

Rest of month:

  • Assemble and annotate 4 transcriptomes we received data for (Day 12 infected, Day 12 uninfected, Day 26 infected, and Day 26 unninfected)
  • Have some results ready to present for GSS (November 14th – my talk is at 1:30pm)
  • Keep updating crab paper

Oyster project

  • FINISH PAPER!! October ended way sooner than I was expecting. Finish it ASAP so that I can send it off somewhere (Scientific Reports?) and focus on crab project
  • Clean up repo

MS SAFS Duties

  • Once crab extractions and sample pooling is done, finish MS proposal
  • Submit MS proposal (Chapter 1: Oyster; Chapter 2: Crab) and Plan of Study to school

One Health Certificate

  • Come up with plan of when I’ll do my capstone (likely Spring quarter)

Other:

Make sure I have a TA position lined up for Winter 2020 (waiting to hear back for one)

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Sam’s Notebook: Data Wrangling – Splitting BAM by Size for Upload to OSF

We’re in the process of organizing files for a manuscript dealing with the geoduck genome assembly/annotation we’ve done. As part of that, we need the Stringtie BAM file that was used with GenSAS for Pgenerosa_v074 annotation to upload to the Open Science Foundation repository for this project. Unfortunately, at 73GB, the file far exceeds the individual file size limit for OSF (5GB). So, I split it into 5GB chunks. See the following notebook for deets:

Jupyter Notebook (GitHub):

TL;DR:

  1. Use Bash command split to split the file into desired chunk sizes
  2. Reassemble chunks into full size BAM using the Bash cat command.
  3. Run md5sum on original BAM and reassembled BAM to confirm the two files are the same.

Grace’s Notebook: RNA extractions – more day 12 – RNA in 23 / 24

Today I extracted RNA from 24 more samples from Day 12. Mix of infection status and temperature exposures. There was RNA in 23 out of 24 samples. No obvious mistakes were made, and at the end of the post I provide an updated RNA sample summary in preparation for our crab RNA meeting tomorrow.

Samples extracted today:

FRP trtmnt_tank sample_day infection_status maturity tube_number
6136 ambient 12 0 M 321
6143 ambient 12 0 M 343
6150 ambient 12 0 M 312
6168 ambient 12 1 I 357
6201 ambient 12 1 I 341
6202 ambient 12 1 I 337
6128 cold 12 1 I 231
6140 cold 12 1 I 237
6141 cold 12 1 I 249
6153 cold 12 0 M 209
6157 cold 12 0 M 224
6160 cold 12 0 M 238
6173 cold 12 1 I 210
6174 cold 12 1 I 233
6175 cold 12 0 I 246
6223 warm 12 0 M 270
6228 warm 12 0 I 290
6230 warm 12 0 M 292
6240 warm 12 1 I 362
6243 warm 12 1 I 272
6244 warm 12 1 I 284
6248 warm 12 0 I 378
6253 warm 12 0 I 275
6255 warm 12 1 I 262

Sample preparation and extraction

I did everything the same as I’ve done the past three extractions (1, 2, and 3).

Results:

qubit_tube_conc_ng.ml original_sample_conc_ng.ul sample_vol_ul dilution_factor tube_number extraction_method ul_sample-used elution_vol_ul total-yield_ng
413 41.3 2 100 262 Zymo_microprep 35 15 536.9
298 29.8 2 100 275 Zymo_microprep 35 15 387.4
141 14.1 2 100 378 Zymo_microprep 35 15 183.3
276 27.6 2 100 284 Zymo_microprep 35 15 358.8
94.2 9.42 2 100 272 Zymo_microprep 35 15 122.46
134 13.4 2 100 362 Zymo_microprep 35 15 174.2
172 17.2 2 100 292 Zymo_microprep 35 15 223.6
285 28.5 2 100 290 Zymo_microprep 35 15 370.5
293 29.3 2 100 270 Zymo_microprep 35 15 380.9
207 20.7 2 100 246 Zymo_microprep 35 15 269.1
352 35.2 2 100 233 Zymo_microprep 35 15 457.6
333 33.3 2 100 210 Zymo_microprep 35 15 432.9
380 38 2 100 238 Zymo_microprep 35 15 494
192 19.2 2 100 224 Zymo_microprep 35 15 249.6
243 24.3 2 100 209 Zymo_microprep 35 15 315.9
186 18.6 2 100 249 Zymo_microprep 35 15 241.8
50.6 5.06 2 100 237 Zymo_microprep 35 15 65.78
379 37.9 2 100 231 Zymo_microprep 35 15 492.7
Out of range Out of range 2 100 337 Zymo_microprep 35 15 #VALUE!
179 17.9 2 100 341 Zymo_microprep 35 15 232.7
173 17.3 2 100 357 Zymo_microprep 35 15 224.9
142 14.2 2 100 312 Zymo_microprep 35 15 184.6
77.3 7.73 2 100 343 Zymo_microprep 35 15 100.49
117 11.7 2 100 321 Zymo_microprep 35 15 152.1

Where are they now?

  • The extracted RNA is in a new box in the -80C: Rack 3, Column 4, Row 2.
  • The hemolymph pellets were placed back where I found them in the Day 12 pelleted hemolymph boxes (Rack 14)
  • The DNA from today and the previous days were transferred to boxes that I labeled and they are on the top shelf in FTR 209 -20C freezer.

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