Concentrating Gametogenesis Acropora pulchra RNA for De Novo Transcriptome Assembly

Goal:

Use Acropora pulchra samples from Mo’orea, French Polynesia, sampled on January 15th 2022 from the north shore backreef site Mahana (17°29’13.9”S 149°53’14.7”W) to create a de novo transcriptome for A. pulchra. Extracted samples (August 23rd 2022) where concentrated using the Zymo RNA Clean and Concentrate Kit on April 25th 2023 at URI.

Sample Collection

Samples were collected and preserved in DNA-RNA shield in Mo’orea, French Polynesia following the Sample Collection Protocol. Initial samples had 1-2 mL of DNA RNA shield added.

Sample Information

January 2022 samples were brought back with CITES permit 20220819 and stored at URI. Samples DNA/RNA was extracted on August 23rd 2022 following this exact protocol.

Sample IDs

11 January 2022 samples used for transcriptome

timepoint tube.number collection.date colony.ID uL taken
January 2022 418 20220115 ACR-418 300
January 2022 422 20220115 ACR-422 300
January 2022 428 20220115 ACR-428 300
January 2022 432 20220115 ACR-432 300
January 2022 438 20220115 ACR-438 300
January 2022 457 20220115 ACR-457 300
January 2022 458 20220115 ACR-458 300
January 2022 459 20220115 ACR-459 300
January 2022 460 20220115 ACR-460 300
January 2022 464 20220115 ACR-464 300
January 2022 465 20220115 ACR-465 300

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DNA/RNA Quantity - 11 January 2022 Samples

tube.number colony.ID qubit.reading.1 qubit.reading.2 quibit.average sample.type timepoint
DNA_standard_1 NA 200.58 NA 200.58 DNA.STD NA
DNA_standard_2 NA 24151 NA 24151 DNA.STD NA
1 ACR-418 25.6 25.4 25.5 DNA JANUARY
2 ACR-422 24 23.4 23.7 DNA JANUARY
3 ACR-428 19.8 19.3 19.55 DNA JANUARY
5 ACR-432 15.1 14.8 14.95 DNA JANUARY
6 ACR-438 25 24.4 24.7 DNA JANUARY
7 ACR-457 27.6 27 27.3 DNA JANUARY
8 ACR-458 29.8 29 29.4 DNA JANUARY
9 ACR-459 13.1 13 13.05 DNA JANUARY
10 ACR-460 15.6 15.1 15.35 DNA JANUARY
11 ACR-464 19.2 18.8 19 DNA JANUARY
12 ACR-465 14 13.5 13.75 DNA JANUARY
RNA_standard_1 NA 422.43 NA 422.43 RNA.STD NA
RNA_standard_2 NA 9892.36 NA 9892.36 RNA.STD NA
1 ACR-418 31.6 31.6 31.6 RNA JANUARY
2 ACR-422 21.6 22 21.8 RNA JANUARY
3 ACR-428 27.6 27.6 27.6 RNA JANUARY
5 ACR-432 16.8 16.4 16.6 RNA JANUARY
6 ACR-438 15.2 14.4 14.8 RNA JANUARY
7 ACR-457 18.4 18.2 18.3 RNA JANUARY
8 ACR-458 15.2 15.2 15.2 RNA JANUARY
9 ACR-459 17.4 16.6 17 RNA JANUARY
10 ACR-460 20.4 20.6 20.5 RNA JANUARY
11 ACR-464 13.8 13.8 13.8 RNA JANUARY
12 ACR-465 13.2 13 13.1 RNA JANUARY

Clean and Concentrate Protocol

For our desired sequence volume to submit to Genewiz for RNAseq for transcriptome analyses, we needed one sample with >50ng/uL. Using the 11 samples above to get a diverse range of genotypes and RNA form multiple individuals, we followed the Zymo RNA Clean Concentrate Protocol based on the volume for each tube for the Zymo Clean and Concentrate kit, eluting in 25 µL of DNAse/RNAse free water.

10 uL of each sample (10 uL * 11 samples = 110 uL) was added to a 1.5 mL microcentrifuge tube

Base volume: 110 uL

  1. Since each sample is 110 uL, add 220 uL (2 x 110) of RNA binding buffer.
  2. Add an equal volume (330 uL (3 x 110)) of 100% ethanol and mix.
  3. Transfer the sample to the Zymo-Spin™ IC Column in a Collection Tube and centrifuge. Discard the flow-through.
  4. Add 400 µl RNA Prep Buffer to the column and centrifuge. Discard the flow-through.
  5. Add 700 µl RNA Wash Buffer to the column and centrifuge. Discard the flow-through.
  6. Add 400 µl RNA Wash Buffer to the column and centrifuge for 1 minute to ensure complete removal of the wash buffer. Carefully, transfer the column into a RNase-free tube (not provided).
  7. Add 25 µl DNase/RNase-Free Water directly to the column matrix and centrifuge.

Qubit Results

  • Used High Sensitivity range dsDNA and RNA Qubit Protocol
  • The concentrated sample was read twice, the standards were only read once

RNA Standards: 418.23 (S1) & 8244.21 (S2)

Concentrating all sample

colony_id RNA_QBIT_AVG Volume in Tube currently Amount RNA in whole tube
ACRP 43.2 24 1036.8
Written on April 25, 2023