Becker DNA RNA Extractions

Danielle Becker Coral Samples Processing January 2020

DNA RNA Extractions

20200123 Test DNA RNA extraction round for Danielle Becker coral samples.

DNA/RNA Extractions from Danielle Becker’s Pocillopora coral samples using the following protocol: Zymo Duet RNA DNA Extraction Protocol and modified adult fragment tissue preparation steps described below:

  • Half of coral fragment clipped and 500 μl of DNA/RNA shield from sample tube placed in tube with 2 mL 0.5 mm glass beads.
  • 40 seconds at 20 Hz in the Tissue Lyser to remove tissue from coral skeleton.
  • Supernatant removed and placed in 1.5 mL microcentrifuge tube for a stock solution (~700 μl).
  • 500 μl of tissue homogenate, 50 μl of Proteinase K Digestion buffer (1:10 ratio with tissue homogenate volume), and 25 μl of Proteinase K added to a new 1.5 mL microcentrifuge tube.
  • 575 μl of Lysis Buffer added (1:1 ratio to tissue, Proteinase K Digestion buffer, and Proteinase K volume)
  • 1,150 μl of 100% molecular grade ethanol added to tissue for RNA portion of the protocol (1:1 ratio)
  • 50 μl warmed Tris added to yellow spin column and incubated at room temperature for 5 minutes before centrifuged, and repeated for a total of 100 μl DNA.
  • 50 μl warmed DNA RNA free water added to green spin column and incubated at room temperature for 5 minutes before centrifuged, and repeated for a total of 100 μl RNA.

Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.

Broad Range: DNA Standard 1: 141.84 ng/μl
DNA Standard 2: 18,643.22 ng/μl

High Sensitivity: RNA Standard 1: 50.93 ng/μl
RNA Standard 2: 963.31 ng/μl

Coral ID DNA 1 DNA 2 Average DNA RNA 1 RNA 2 Average RNA RIN
E9 11.4 11.4 11.4 21.2 21.2 21.2 9.2
E11 6.84 6.8 6.82 9.82 9.96 9.89 8.8
C31 15.5 15.4 15.45 10.2 10.2 10.2 7.7

Link to 20200123 TapeStation report

Gel Electrophoresis Results:

20200123 Extractions

20200127 DNA RNA extraction round for Danielle Becker coral samples.

DNA/RNA Extractions from Danielle Becker’s Pocillopora coral samples using the following protocol: Zymo Duet RNA DNA Extraction Protocol and modified adult fragment tissue preparation steps described below:

  • Whole coral clipping and 700 μl of DNA/RNA shield from sample tube placed in tube with 2 mL 0.5 mm glass beads and 300 μl fresh DNA/RNA shield.
  • 40 seconds at 20 Hz in the Tissue Lyser to remove tissue from coral skeleton.
  • Supernatant removed and placed in 1.5 mL microcentrifuge tube for a stock solution (~700 μl).
  • 500 μl of tissue homogenate, 50 μl of Proteinase K Digestion buffer (1:10 ratio with tissue homogenate volume), and 25 μl of Proteinase K added to a new 1.5 mL microcentrifuge tube.
  • 575 μl of Lysis Buffer added (1:1 ratio to tissue, Proteinase K Digestion buffer, and Proteinase K volume)
  • 1,150 μl of 100% molecular grade ethanol added to tissue for RNA portion of the protocol (1:1 ratio)
  • 50 μl warmed Tris added to yellow spin column and incubated at room temperature for 5 minutes before centrifuged, and repeated for a total of 100 μl DNA.
  • 50 μl warmed DNA RNA free water added to green spin column and incubated at room temperature for 5 minutes before centrifuged, and repeated for a total of 100 μl RNA.

Half of RNA flow-through from samples C30, E4, and E10 thrown out accidentally. 575 μl of sample (original sample, Proteinase K Digestion Buffer, and Proteinase K) and 575 μl of 100% molecular grade ethanol used. Spun down twice in total. All other samples followed 1,150 μl of sample and 1,150 μl of 100% ethanol protocol steps.

Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.

Broad Range: DNA Standard 1: 142.77 ng/μl
DNA Standard 2: 20,503.32 ng/μl

High Sensitivity: RNA Standard 1: 51.31 ng/μl
RNA Standard 2: 1,010.38 ng/μl

Coral ID DNA 1 DNA 2 Average DNA RNA 1 RNA 2 Average RNA RIN
C30 6.64 6.6 6.62 ** ** ** **
E4 12 12 12 4.6 4.6 4.6 **
C25 19.7 19.7 19.7 18.3 18.3 18.3 9.2
C32 7.72 7.68 7.7 ** ** ** **
E6 8.34 8.32 8.33 8.38 8.44 8.41 9.1
C29 7.38 7.36 7.37 ** ** ** **
E1 6.08 6.04 6.06 ** ** ** **
E3 23.8 23.8 23.8 ** ** ** **
E16 12.4 12.4 12.4 7.76 7.88 7.82 9
E8 4.68 4.66 4.67 ** ** ** **
E15 6.56 6.52 6.54 ** ** ** **
C21 6.2 6.18 6.19 ** ** ** **
C22 14.1 14 14.05 ** ** ** **
E14 9.58 9.52 9.55 ** ** ** **
E10 9.86 9.82 9.84 4.4 4.4 4.4 **

Link to 20200127 TapeStation report

Gel Electrophoresis Results:

20200127 Extractions

20200128 DNA RNA extraction round for Danielle Becker coral samples.

DNA/RNA Extractions from Danielle Becker’s Pocillopora coral samples using the following protocol: Zymo Duet RNA DNA Extraction Protocol and modified adult fragment tissue preparation steps described below:

  • Whole coral clipping and 700 μl of DNA/RNA shield from sample tube placed in tube with 2 mL 0.5 mm glass beads and 300 μl fresh DNA/RNA shield.
  • 40 seconds at 20 Hz in the Tissue Lyser to remove tissue from coral skeleton.
  • Supernatant removed and placed in 1.5 mL microcentrifuge tube for a stock solution (~700 μl).
  • 600 μl of tissue homogenate, 60 μl of Proteinase K Digestion buffer (1:10 ratio with tissue homogenate volume), and 30 μl of Proteinase K added to a new 1.5 mL microcentrifuge tube.
  • 690 μl of Lysis Buffer added (1:1 ratio to tissue, Proteinase K Digestion buffer, and Proteinase K volume)
  • 1,380 μl of 100% molecular grade ethanol added to tissue for RNA portion of the protocol (1:1 ratio)
  • 50 μl warmed Tris added to yellow spin column and incubated at room temperature for 5 minutes before centrifuged, and repeated for a total of 100 μl DNA.
  • 50 μl warmed DNA RNA free water added to green spin column and incubated at room temperature for 5 minutes before centrifuged, and repeated for a total of 100 μl RNA.

Post tissue lyser: 1,000 μl DNA RNA Shield added to samples E5, C17, and E7 because there might be enough tissue on the skeleton to try another extraction if necessary. These samples had larger clippings.

Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.

Broad Range: DNA Standard 1: 162.98 ng/μl
DNA Standard 2: 23,670.63 ng/μl

High Sensitivity: RNA Standard 1: 51.25 ng/μl
RNA Standard 2: 995.63 ng/μl

Coral ID DNA 1 DNA 2 Average DNA RNA 1 RNA 2 Average RNA RIN
C17 19.1 18.3 18.7 23 23 23 9.3
E2 11.3 11.3 11.3 11 11.1 11.05 8.8
C18 14.7 14.7 14.7 17.5 17.5 17.5 9.2
C19 9.2 9.16 9.18 8.52 8.46 8.49 8.2
E5 17.7 17.7 17.7 11.8 11.9 11.85 8.3
E13 15.9 15.8 15.85 15.1 15 15.05 9.6

Link to 20200128 TapeStation report

Gel Electrophoresis Results:

20200128 Extractions

Photos of clipping size to compare to qubit values:

Control 17:
C17

Enriched 2:
E2

Control 18:
C18

Control 19:
C19

Enriched 5:
E5

Enriched 13:
E13

Enriched 7:
E7

20200129 DNA RNA extraction round for Danielle Becker coral samples.

DNA/RNA Extractions from Danielle Becker’s Pocillopora coral samples using the following protocol: Zymo Duet RNA DNA Extraction Protocol and modified adult fragment tissue preparation steps described below:

  • Whole coral clipping and 700 μl of DNA/RNA shield from sample tube placed in tube with 2 mL 0.5 mm glass beads and 300 μl fresh DNA/RNA shield.
  • 40 seconds at 20 Hz in the Tissue Lyser to remove tissue from coral skeleton.
  • Supernatant removed and placed in 1.5 mL microcentrifuge tube for a stock solution (~700 μl).
  • 600 μl of tissue homogenate, 60 μl of Proteinase K Digestion buffer (1:10 ratio with tissue homogenate volume), and 30 μl of Proteinase K added to a new 1.5 mL microcentrifuge tube.
  • 690 μl of Lysis Buffer added (1:1 ratio to tissue, Proteinase K Digestion buffer, and Proteinase K volume)
  • 1,380 μl of 100% molecular grade ethanol added to tissue for RNA portion of the protocol (1:1 ratio)
  • 50 μl warmed Tris added to yellow spin column and incubated at room temperature for 5 minutes before centrifuged, and repeated for a total of 100 μl DNA.
  • 50 μl warmed DNA RNA free water added to green spin column and incubated at room temperature for 5 minutes before centrifuged, and repeated for a total of 100 μl RNA.

Post tissue lyser: 1,000 μl DNA RNA Shield added to larger sample clippings because there might be enough tissue on the skeleton to try another extraction if necessary. These samples had larger clippings.

1,830 μl of 100% ethanol accidentally added to all of the RNA samples instead of 1,380 μl.

Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.

Broad Range: DNA Standard 1: 155.65 ng/μl
DNA Standard 2: 22,158.47 ng/μl

High Sensitivity: RNA Standard 1: 52.72 ng/μl
RNA Standard 2: 1,037.95 ng/μl

Date Coral ID DNA 1 DNA 2 Average DNA (ng_uL) RNA 1 RNA 2 Average RNA (ng_uL) RIN
20200129 E12 17.6 17.5 17.55 13.9 13.9 13.9 9.1
20200129 C20 21 20.8 20.9 11.7 11.7 11.7 8.2
20200129 C23 10.9 10.9 10.9 6.4 6.36 6.38 7.4
20200129 C24 12.7 12.7 12.7 13.5 13.6 13.55 9.3
20200129 C26 7.94 7.92 7.93 ** ** ** 6
20200129 C27 9.18 9.16 9.17 ** ** ** **
20200129 C28 22.2 22.2 22.2 12.2 12.2 12.2 8.9

Link to 20200129 TapeStation report

Gel Electrophoresis Results:

20200129 Extractions

Summary of DNA RNA Extraction Results

Date Coral ID DNA 1 DNA 2 Average DNA (ng_uL) RNA 1 RNA 2 Average RNA (ng_uL) RIN
20200127 E1 6.08 6.04 6.06 ** ** ** **
20200128 E2 11.3 11.3 11.3 11 11.1 11.05 8.8
20200127 E3 23.8 23.8 23.8 ** ** ** **
20200127 E4 12 12 12 4.6 4.6 4.6 **
20200128 E5 17.7 17.7 17.7 11.8 11.9 11.85 8.3
20200127 E6 8.34 8.32 8.33 8.38 8.44 8.41 9.1
20200128 E7 18.9 18.9 18.9 15.6 15.5 15.55 8.8
20200127 E8 4.68 4.66 4.67 ** ** ** **
20200123 E9 11.4 11.4 11.4 21.2 21.2 21.2 9.2
20200127 E10 9.86 9.82 9.84 4.4 4.4 4.4 **
20200123 E11 6.84 6.8 6.82 9.82 9.96 9.89 8.8
20200129 E12 17.6 17.5 17.55 13.9 13.9 13.9 9.1
20200128 E13 15.9 15.8 15.85 15.1 15 15.05 9.6
20200127 E14 9.58 9.52 9.55 ** ** ** **
20200127 E15 6.56 6.52 6.54 ** ** ** **
20200127 E16 12.4 12.4 12.4 7.76 7.88 7.82 9
20200128 C17 19.1 18.3 18.7 23 23 23 9.3
20200128 C18 14.7 14.7 14.7 17.5 17.5 17.5 9.2
20200128 C19 9.2 9.16 9.18 8.52 8.46 8.49 8.2
20200129 C20 21 20.8 20.9 11.7 11.7 11.7 8.2
20200127 C21 6.2 6.18 6.19 ** ** ** **
20200127 C22 14.1 14 14.05 ** ** ** **
20200129 C23 10.9 10.9 10.9 6.4 6.36 6.38 7.4
20200129 C24 12.7 12.7 12.7 13.5 13.6 13.55 9.3
20200127 C25 19.7 19.7 19.7 18.3 18.3 18.3 9.2
20200129 C26 7.94 7.92 7.93 ** ** ** 6
20200129 C27 9.18 9.16 9.17 ** ** ** **
20200129 C28 22.2 22.2 22.2 12.2 12.2 12.2 8.9
20200127 C29 7.38 7.36 7.37 ** ** ** **
20200127 C30 6.64 6.6 6.62 ** ** ** **
20200123 C31 15.5 15.4 15.45 10.2 10.2 10.2 7.7
20200127 C32 7.72 7.68 7.7 ** ** ** **

ITS2 Sequencing

The Internal Transcribed Spacer 2 (ITS2) gene was isolated from all DNA samples above using the following Putnam Lab ITS2 Sequencing Protocol.

Dilution aliquot calculations:

Amplicon ID Coral ID Extraction Date Hard DNA (ng_ul) DNA for dilution (ul) Water for dilution (ul) Dilution Strip Tube #
1 E9 20200127 11.4 2.89 7.11 1
2 E11 20200128 6.82 4.84 5.16 1
3 C31 20200127 15.45 2.14 7.86 1
4 C30 20200127 6.62 4.98 5.02 1
5 E4 20200128 12 2.75 7.25 1
6 C25 20200127 19.7 1.68 8.32 1
7 C32 20200128 7.7 4.29 5.71 1
8 E6 20200127 8.33 3.96 6.04 1
9 C29 20200123 7.37 4.48 5.52 2
10 E1 20200127 6.06 5.45 4.55 2
11 E3 20200123 23.8 1.39 8.61 2
12 E16 20200129 12.4 2.66 7.34 2
13 E8 20200128 4.67 7.07 2.93 2
14 E15 20200127 6.54 5.05 4.95 2
15 C21 20200127 6.19 5.33 4.67 2
16 C22 20200127 14.05 2.35 7.65 2
17 E14 20200128 9.55 3.46 6.54 3
18 E10 20200128 9.84 3.35 6.65 3
19 C17 20200128 18.7 1.76 8.24 3
20 E2 20200129 11.3 2.92 7.08 3
21 C18 20200127 14.7 2.24 7.76 3
22 C19 20200127 9.18 3.59 6.41 3
23 E5 20200129 17.7 1.86 8.14 3
24 E13 20200129 15.85 2.08 7.92 3
25 E7 20200127 18.9 1.75 8.25 4
26 E12 20200129 17.55 1.88 8.12 4
27 C27 20200129 9.17 3.6 6.4 4
28 C24 20200129 12.7 2.6 7.4 4
29 C23 20200127 10.9 3.03 6.97 4
30 C20 20200127 20.9 1.58 8.42 4
31 C28 20200123 22.2 1.49 8.51 4
32 C26 20200127 7.93 4.16 5.84 4
33 MIX   18.2 4.57 20.43 5
34 NEG CON   12.75 6.53 18.47 5

96-well plate maps:

Plate 1:

NA 1 2 3 4 5 6 7 8 9 10 11 12
A 1 1 1 9 9 9 17 17 17 NEGCON NEGCON NEGCON
B 2 2 2 10 10 10 18 18 18      
C 3 3 3 11 11 11 19 19 19      
D 4 4 4 12 12 12 20 20 20      
E 5 5 5 13 13 13 21 21 21      
F 6 6 6 14 14 14 22 22 22      
G 7 7 7 15 15 15 23 23 23      
H 8 8 8 16 16 16 24 24 24      

Plate 2:

NA 1 2 3 4 5 6 7 8 9 10 11 12
A 25 25 25 33 33 33            
B 26 26 26 NEGCON NEGCON NEGCON            
C 27 27 27                  
D 28 28 28                  
E 29 29 29                  
F 30 30 30                  
G 31 31 31                  
H 32 32 32                  

Gel Electrophoresis Image:

Becker

Sequencing center ID:

DataID AmpliconID SampleName AmpliconGene StripTube Number
HP401 1 E9 ITS2 1
HP402 2 E11 ITS2 1
HP403 3 C31 ITS2 1
HP404 4 C30 ITS2 1
HP405 5 E4 ITS2 1
HP406 6 C25 ITS2 1
HP407 7 C32 ITS2 1
HP408 8 E6 ITS2 1
HP409 9 C29 ITS2 2
HP410 10 E1 ITS2 2
HP411 11 E3 ITS2 2
HP412 12 E16 ITS2 2
HP413 13 E8 ITS2 2
HP414 14 E15 ITS2 2
HP415 15 C21 ITS2 2
HP416 16 C22 ITS2 2
HP417 17 E14 ITS2 3
HP418 18 E10 ITS2 3
HP419 19 C17 ITS2 3
HP420 20 E2 ITS2 3
HP421 21 C18 ITS2 3
HP422 22 C19 ITS2 3
HP423 23 E5 ITS2 3
HP424 24 E13 ITS2 3
HP425 25 E7 ITS2 4
HP426 26 E12 ITS2 4
HP427 27 C27 ITS2 4
HP428 28 C24 ITS2 4
HP429 29 C23 ITS2 4
HP430 30 C20 ITS2 4
HP431 31 C28 ITS2 4
HP432 32 C26 ITS2 4
HP433 33 MIX ITS2 5
HP434 34 NEG CON ITS2 5

Re-extracting the samples with no RNA yield

Extractions from re-bead beating coral fragments + 300 uL of original shield

DNA/RNA Extractions from Danielle Becker’s Pocillopora coral samples using the following protocol: Zymo Duet RNA DNA Extraction Protocol and modified adult fragment tissue preparation steps described below:

Tissue fragment preparation:

  • Thawing the bead tube that contains the fragments and beads from the original extraction
  • Adding 300 uL of the original DNA RNA shield and 700 uL of new DNA RNA shield
  • 40 seconds at 20 Hz in the Tissue Lyser to remove reamining tissue from coral skeleton
  • Supernatant removed and placed in 1.5 mL microcentrifuge tube for the extraction protocol (Zymo link above) with the following modifications.
  • No stock solution leftover from 20200901 extraction. ~300 uL of stock solution remains from the original extraction in case 20200901 extraction fails.

Extraction modification steps:

  • 700 μl of tissue homogenate, 70 μl of Proteinase K Digestion buffer (1:10 ratio with tissue homogenate volume), and 35 μl of Proteinase K added to a new 1.5 mL microcentrifuge tube.
  • 805 μl of Lysis Buffer added (1:1 ratio to tissue, Proteinase K Digestion buffer, and Proteinase K volume)
  • 1,610 μl of 100% molecular grade ethanol added to tissue for RNA portion of the protocol (1:1 ratio)

20200901 notes:

  • No incubation step used for final DNA elution steps (accidentally skipped). Lower DNA yield from these samples but should still be usable.

20200907 notes:

  • Supernatant is very clear, hardly any tissue on these fragment pieces. Worried about not getting enough genomic pool from the coral. Starting to think the original stock would better biologically even though there is only 300 uL leftover.
  • C32 did not have any original DNA RNA shield left. 1,000 uL of new RNA DNA shield used.

Qubit and TapeStation Results

Extractions from coral fragment pieces and original DNA RNA shield

Date Coral ID DNA 1 DNA 2 Average DNA (ng_uL) RNA 1 RNA 2 Average RNA (ng_uL) RIN Pass?
20200901 E3 10.9 10.8 10.85 16.8 16.8 16.8 8.3 Yes
20200901 E8 3 2.98 2.99 6.96 7 6.98 ** No
20200901 E14 7.46 7.4 7.43 15.5 15.5 15.5 8.9 Yes
20200901 C22 7.78 7.76 7.77 23.6 23.6 23.6 9.4 Yes
20200907 E1 9.5 9.46 9.48 8.74 8.72 8.73 ** Re do tape?
20200907 E15 3.74 3.68 3.71 5.54 5.42 5.48 ** No
20200907 C21 2.66 2.64 2.65 7.72 7.82 7.77 8.8 Yes
20200907 C26 4.78 4.74 4.76 5.88 5.9 5.89 ** No
20200907 C27 3.52 3.5 3.51 4.2 4.4 4.3 ** No
20200907 C29 4.5 4.48 4.49 6.74 6.72 6.73 ** No
20200907 C30 5.34 5.28 5.31 9.3 9.32 9.31 8.6 Yes
20200907 C32 3.02 3.02 3.02 ** ** ** ** No
20200915 E4 5.2 4.96 5.08 15.1 15.1 15.1 9.3 Yes
20200915 E10 11.4 11.3 11.35 23.2 23.2 8 8 Yes

20200901 Extractions: DNA BR Standard 1: 199.24 ng/μl
DNA BR Standard 2: 22,582.06 ng/μl
RNA HS Standard 1: 49.18 ng/μl
RNA HS Standard 2: 907.40 ng/μl

20200907 Extractions: DNA BR Standard 1: 192.94 ng/μl
DNA BR Standard 2: 21,521.22 ng/μl
RNA HS Standard 1: 50.71 ng/μl
RNA HS Standard 2: 952.91 ng/μl

2020915 Extractions:
DNA BR Standard 1: 168.02 ng/μl
DNA BR Standard 2: 19,640.60 ng/μl
RNA HS Standard 1: 50.14 ng/μl
RNA HS Standard 2: 924.90 ng/μl

20200901 E3, E8, E14, C22 TapeStation Results

20200901 E3, E8, E14, C22 Gel Image gel

20200908 TapeStation Results

20200908 Gel Image gel2

20200915 TapeStation

20200915 Gel image

gel3

Although I did get RNA from this re-extraction round, I am now thinking that doing extractions on the original stock (only 300 uL) would be better biologically. The original stock would have more coral tissue and is darker in color than what I got from beating the coral fragments again. Only issue is that I wasn’t originally able to get enough from 300 uL on the first time around which is why I hadn’t done re-extractions on those yet. If this works, I would take the RNA and DNA from the stock instead of what was done on 20200901 and 20200907.

Next steps: 20200912 Extractions on the 300 uL stock from all samples. Two rounds of 6 samples. This is the last shot on this batch of corals. If still not satisfied with results, then consider doing cites for the batch in Mo’orea.

Extractions from 300 uL original stock solution from the first time bead beating the fragments

20200911:
2 rounds of extractions: 1.) E8, C22, E14, E8, C29, E15 and 2.) C30, C21, E1, C27, C26, C32

Stock is from original Tissue Lyser run, go straight into ProK Buffer and ProK step. (300 uL sample, 30 uL Prok Buffer, 15 uL ProK).

20200914 Qubit, TapeStation DNA BR Standard 1: 194.38 ng/μl
DNA BR Standard 2: 21,798.00 ng/μl
RNA HS Standard 1: 49.37 ng/μl
RNA HS Standard 2: 941.70 ng/μl

Date Coral ID DNA 1 DNA 2 Average DNA (ng_uL) RNA 1 RNA 2 Average RNA (ng_uL) RIN Pass?
20200911 E3 8.6 8.56 8.58 7.28 7.36 7.32 ** No
20200911 E8 ** ** ** ** ** ** ** No
20200911 E14 5.04 5.02 5.03 5.12 5.1 5.11 ** No
20200911 C22 7.9 7.86 7.88 10.7 10.7 10.7 8.9 Yes
20200911 E1 2.66 2.64 2.65 5.18 5.26 5.22 ** No
20200911 E15 ** ** ** ** ** ** ** No
20200911 C21 2.44 2.42 2.43 4.6 4.6 4.6 ** No
20200911 C26 ** ** ** ** ** ** ** No
20200911 C27 ** ** ** ** ** ** ** No
20200911 C29 2.56 2.56 2.56 5.14 5.08 5.11 ** No
20200911 C30 2.42 2.4 2.41 6.04 6.04 6.04 ** No
20200911 C32 2.5 2.46 2.48 ** ** ** ** No

20200914 TapeStation Results

No gel image for these extractions because GelGreen was accidentally left out of the gel. Not re-done because Qubit values were low anyways.

This method didn’t work as well as I had hoped. Could be that the stock de-thawing caused degradation of DNA RNA? Use the extractions done with re-bead beating the coral fragments.

Just to double check:
Re-qubit C26 RNA from January 2019.
RNA HS Standard 1: 49.57 ng/μl
RNA HS Standard 2: 964.11 ng/μl Value 1: 5.04 ng/uL Value 2: 5.04 ng/uL

Re-TapeStation E1 from January 2019.
Value returned as too low to detect. TapeStation Report

Removed E1 and C26 from the final list to send to sequencing.

20200921:
The below will be combined and re-qubit prior to sequencing.

Date Coral ID DNA 1 DNA 2 Average DNA (ng_uL) RNA 1 RNA 2 Average RNA (ng_uL) RIN Pass? Qualitative RIN by HP  
20200907 E1 9.5 9.46 9.48 8.74 8.72 8.73 ** No Fine Combine
20200911 E1 2.66 2.64 2.65 5.18 5.26 5.22 ** No Fine Combine
20200129 C26 7.94 7.92 7.93 5.04 5.04 5.04 6 No OK Combine
20200907 C26 4.78 4.74 4.76 5.88 5.9 5.89 ** No OK Combine
20200907 C29 4.5 4.48 4.49 6.74 6.72 6.73 ** No Fine Combine
20200911 C29 2.56 2.56 2.56 5.14 5.08 5.11 ** No Fine Combine
20200907 C32 3.02 3.02 3.02 ** ** ** ** No Fine, but low Combine
20200911 C32 2.5 2.46 2.48 ** ** ** ** No Fine, but low Combine

New qubit values:

Date Coral ID DNA 1 DNA 2 Average DNA (ng_uL) RNA 1 RNA 2 Average RNA (ng_uL) RIN Pass? Qualitative RIN by HP
20200922 E1 7.6 7.5 7.55 9.52 9.6 9.56 8.6 Yes Fine
20200922 C26 7.52 7.44 7.48 6.3 6.34 6.32 ** Yes Fine
20200922 C29 4.84 4.78 4.81 6.44 6.42 6.43 ** Yes Fine
20200922 C32 3.6 3.48 3.54 4.4 ** 4.4 ** Yes Fine

Combined DNA on gel 20200923:

gel1

Final Extraction Values

20200916 final thoughts: Some of the coral had enough tissue on the fragments to be re-bead beated successfully. There was also likely DNA and RNA in the original shield volume. I now think this is the better option biologically because freeze-thawing stock or tissue homogenate more than once degrades the DNA and RNA in the homogenate. Which you can see based on the results of the second type of re-extractions based on the stock, and this also happened to Kevin (labmate) with his 2017 vs 2018 tissue homogenates.

Dates in January 2020 are original extraction values. Dates in September 2020 are re-extraction values.

Below are the samples that have passed DNA and RNA quality and quantity tested post extraction. All fragments are together in a box in the new, blue -80C freezer.

Date Coral ID DNA 1 DNA 2 Average DNA (ng_uL) RNA 1 RNA 2 Average RNA (ng_uL) RIN Pass? Qualitative RIN by HP
20200128 C17 19.1 18.3 18.7 23 23 23 9.3 Yes  
20200128 C18 14.7 14.7 14.7 17.5 17.5 17.5 9.2 Yes  
20200128 C19 9.2 9.16 9.18 8.52 8.46 8.49 8.2 Yes  
20200129 C20 21 20.8 20.9 11.7 11.7 11.7 8.2 Yes  
20200907 C21 2.66 2.64 2.65 7.72 7.82 7.77 8.8 Yes  
20200901 C22 7.78 7.76 7.77 23.6 23.6 23.6 9.4 Yes  
20200129 C23 10.9 10.9 10.9 6.4 6.36 6.38 7.4 Yes  
20200129 C24 12.7 12.7 12.7 13.5 13.6 13.55 9.3 Yes  
20200127 C25 19.7 19.7 19.7 18.3 18.3 18.3 9.2 Yes  
20200922 C26 7.52 7.44 7.48 6.3 6.34 6.32 ** Yes Fine
20200907 C27 3.52 3.5 3.51 4.2 4.4 4.3 ** Yes Fine
20200129 C28 22.2 22.2 22.2 12.2 12.2 12.2 8.9 Yes  
20200922 C29 4.84 4.78 4.81 6.44 6.42 6.43 ** Yes Fine
20200907 C30 5.34 5.28 5.31 9.3 9.32 9.31 8.6 Yes  
20200123 C31 15.5 15.4 15.45 10.2 10.2 10.2 7.7 Yes  
20200922 C32 3.6 3.48 3.54 4.4 *** 4.4 ** Yes Fine
20200922 E1 7.6 7.5 7.55 9.52 9.6 9.56 8.6 Yes Fine
20200128 E2 11.3 11.3 11.3 11 11.1 11.05 8.8 Yes  
20200901 E3 10.9 10.8 10.85 16.8 16.8 16.8 8.3 Yes  
20200915 E4 5.2 4.96 5.08 15.1 15.1 15.1 9.3 Yes  
20200128 E5 17.7 17.7 17.7 11.8 11.9 11.85 8.3 Yes  
20200127 E6 8.34 8.32 8.33 8.38 8.44 8.41 9.1 Yes  
20200128 E7 18.9 18.9 18.9 15.6 15.5 15.55 8.8 Yes  
20200901 E8 3 2.98 2.99 6.96 7 6.98 ** Yes Fine
20200123 E9 11.4 11.4 11.4 21.2 21.2 21.2 9.2 Yes  
20200915 E10 11.4 11.3 11.35 23.2 23.2 8 8 Yes  
20200123 E11 6.84 6.8 6.82 9.82 9.96 9.89 8.8 Yes  
20200129 E12 17.6 17.5 17.55 13.9 13.9 13.9 9.1 Yes  
20200128 E13 15.9 15.8 15.85 15.1 15 15.05 9.6 Yes  
20200901 E14 7.46 7.4 7.43 15.5 15.5 15.5 8.9 Yes  
20200907 E15 3.74 3.68 3.71 5.54 5.42 5.48 ** Yes Fine
20200127 E16 12.4 12.4 12.4 7.76 7.88 7.82 9 Yes  

Labeled tubes in freezer

  • All four boxes contain saved material from the extractions. Once DNA and RNA is back from sequencing and backed up in several places, we can throw away most of the contents of the three white boxes. There are a couple of tubes with coral fragments that can be saved but we won’t need the rest.

labeled tubes

  • Tubes are also labeled on the sides with dates of extraction. Match these with the extraction dates in the tables above to find desired DNA tube.

tubes

Google spreadsheet with raw extraction data

mtORF amplication M. Schedl notebook post

  • mtORF and ITS2 amplication was done with DNA from January 2020 extractions. This was completed prior to September 2020 re-extractions for DNA and RNA in the same genomic pool.

RNASeq Prep for Genewiz

Active spreadsheet with the below data.

I chose to send 500 ng because some samples had low quantity values and all samples sent to Genewiz need to have the same quantity of DNA. uL_to_500ng calculated by =500/Qubit. Check mark indicates the full volume needed was added. Two samples did not have enough total sample so that will be less 500 ng; we will send anyway.

Protocol for RNASeq Prep

Date Coral ID RNA 1 RNA 2 Avg_RNA (ng/uL) uL_to_500ng Notes
20200128 C17 23 23 23 21.74 ✓
20200128 C18 17.5 17.5 17.5 28.57 ✓
20200128 C19 8.52 8.46 8.49 58.89 ✓
20200129 C20 11.7 11.7 11.7 42.74 ✓
20200907 C21 7.72 7.82 7.77 64.35 ✓
20200901 C22 23.6 23.6 23.6 21.19 ✓
20200129 C23 6.4 6.36 6.38 78.37 ✓
20200129 C24 13.5 13.6 13.55 36.90 ✓
20200127 C25 18.3 18.3 18.3 27.32 ✓
20200129 C28 12.2 12.2 12.2 40.98 ✓
20200907 C30 9.3 9.32 9.31 53.71 ✓
20200123 C31 10.2 10.2 10.2 49.02 ✓
20200123 E11 9.82 9.96 9.89 50.56 ✓
20200129 E12 13.9 13.9 13.9 35.97 ✓
20200128 E13 15.1 15 15.05 33.22 ✓
20200901 E14 15.5 15.5 15.5 32.26 ✓
20200127 E16 7.76 7.88 7.82 63.94 ✓
20200128 E2 11 11.1 11.05 45.25 ✓
20200901 E3 16.8 16.8 16.8 29.76 ✓
20200915 E4 15.1 15.1 15.1 33.11 ✓
20200128 E5 11.8 11.9 11.85 42.19 ✓
20200127 E6 8.38 8.44 8.41 59.45 ✓
20200128 E7 15.6 15.5 15.55 32.15 ✓
20200123 E9 21.2 21.2 21.2 23.58 ✓
20200915 E10 23.2 23.2 23.2 21.55 ✓
20200901 E8 6.96 7 6.98 71.63 ✓
20200922 E1 9.52 9.6 9.56 52.30 ✓
20200922 C26 6.3 6.34 6.32 79.11 ✓
20200922 C29 6.44 6.42 6.43 77.76 ✓
20200922 C32 4.4 ** 4.4 113.64 ✓; had enough b/c combined 2 elutions
20200907 E15 5.54 5.42 5.48 91.24 84.8 uL added; that was all we had
20200907 C27 4.2 4.4 4.3 116.28 85.6 uL added; that was all we had

20200923 Update: LN2 ordered; waiting for that to arrive prior to shipping. Samples are parafilmed and ready to be sent.

Written on January 23, 2020