October DNA RNA Extractions

20191001 E.S.

DNA/RNA Extractions from Montipora capitata and Pocillopora acuta adult coral fragments from Holobiont Integration Hawaii 2018 project.

Soft and Hard homogenization, and DNA/RNA Extractions followed this protocol. General Zymo Duet DNA/RNA Extraction protocol found here. With the following changes:

• DNA: The first and second elutions were spun down in separate microcentrifuge tubes to compare the quantity and quality between the first and second elution. The first elution had no incubation period (previously 5 minutes) with 10 μl of Tris buffer and the second elution had a 15 minute incubation period (previously 5 minutes) with 100 μl of Tris buffer.
• 10 μl aliquots of DNA and 5 μl aliquots of RNA (instead of 10 μl of each).

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

DNA Standard 1: 163.56 ng/μl
DNA Standard 2: 22,644.53 ng/μl
RNA Standard 1: 402.39 ng/μl
RNA Standard 2: 10,030.67 ng/μl

Timepoint	Species	Coral ID	Extraction ID	Homogenization	DNA Reading 1	DNA Reading 2	Average DNA ng/μl	RNA Reading 1	RNA Reading 2	Average RNA ng/μl	RIN
20181215	Montipora	2511	485	soft	10.2	9.92	10.06	19.8	19.4	19.6	9.2
20181215	Montipora	2511	486	hard	7.62	7.52	7.57	12.2	12	12.1	NA
20181215	Montipora	2188	487	soft	17.7	17.5	17.6	13.4	13.4	13.4	9.2
20181215	Montipora	2188	488	hard	10.3	10.2	10.25	10.2	10.2	10.2	NA
20181215	Pocillopora	2668	489	soft	28.4	28.2	28.3	90.6	90.6	90.6	9
20181215	Pocillopora	2668	490	hard	31.4	31.2	31.3	52.6	52.4	52.5	NA
20181215	Montipora	2302	491	soft	12.3	12.2	12.25	17.6	17.8	17.7	8.7
20181215	Montipora	2302	492	hard	8.74	8.64	8.69	11.2	11.2	11.2	NA
20181215	Montipora	1248	493	soft	15.5	15.4	15.45	14.4	14.4	14.4	**
20181215	Montipora	1248	494	hard	6.46	6.38	6.42	13.8	13.6	13.7	NA

Link to 20191001 TapeStation report, Extractions #485-494

Gel Electrophoresis Results:

20191009 E.S.

DNA/RNA Extractions from Montipora capitata and Pocillopora acuta adult coral fragments from Holobiont Integration Hawaii 2018 project.

Soft and Hard homogenization, and DNA/RNA Extractions followed this protocol. General Zymo Duet DNA/RNA Extraction protocol found here. With the following changes:

• DNA: The first and second elutions were spun down in separate microcentrifuge tubes to compare the quantity and quality between the first and second elution. The first elution had no incubation period (previously 5 minutes) with 10 μl of Tris buffer and the second elution had a 15 minute incubation period (previously 5 minutes) with 100 μl of Tris buffer.
• 10 μl aliquots of DNA and 5 μl aliquots of RNA (instead of 10 μl of each).

DNA 400 μl Wash Buffer step was centrifuged for 30 seconds instead of 2 minutes.

Bleached fragments: P1416 (HTHC), P1596 (HTAC), P2185 (HTHC)

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

DNA Standard 1: 149.92 ng/μl
DNA Standard 2: 20,432.03 ng/μl
RNA Standard 1: 396.96 ng/μl
RNA Standard 2: 9,194.56 ng/μl

Timepoint	Species	Coral ID	Extraction ID	Homogenization	DNA Reading 1	DNA Reading 2	Average DNA ng/μl	RNA Reading 1	RNA Reading 2	Average RNA ng/μl	RIN
20181215	Montipora	2756	495	soft	29	29	29	37.4	37.4	37.4	9.2
20181215	Montipora	2756	496	hard	9	8.96	8.98	15.2	15.2	15.2	NA
20181215	Pocillopora	1416	497	soft	16.8	16.7	16.75	61.2	61.2	61.2	9
20181215	Pocillopora	1416	498	hard	6.98	6.94	6.96	22.6	22.8	22.7	NA
20181215	Pocillopora	2185	499	soft	26.8	26.8	26.8	49.2	49.2	49.2	9.1
20181215	Pocillopora	2185	500	hard	15.9	15.9	15.9	28.2	28.4	28.3	NA
20181215	Montipora	1270	501	soft	42	42	42	39.2	39.4	39.3	8.9
20181215	Montipora	1270	502	hard	24.8	24.6	24.7	24.2	24.2	24.2	NA
20181215	Pocillopora	1596	503	soft	27.8	27.8	27.8	69.8	70	69.9	9.5
20181215	Pocillopora	1596	504	hard	13.1	13.1	13.1	44.4	44.6	44.5	NA
20190112	Montipora	1452	505	soft	32.6	32.6	32.6	35	35	35	9.2
20190112	Montipora	1452	506	hard	22.8	22.8	22.8	**	**	**	NA
20190112	Montipora	2190	507	soft	48	47.8	47.9	12.4	12.4	12.4	9.1
20190112	Montipora	2190	508	hard	20.4	20.4	20.4	23.6	23.8	23.7	NA
20190112	Montipora	1140	509	soft	23.8	23.6	23.7	20.4	20.6	20.5	9.3
20190112	Montipora	1140	510	hard	14.5	14.5	14.5	15.4	15.6	15.5	NA
20190112	Montipora	1632	511	soft	32.4	32.4	32.4	23.2	23.4	23.3	9.4
20190112	Montipora	1632	512	hard	11.1	11.1	11.1	20.8	20.8	20.8	NA
20190112	Montipora	2990	513	soft	10.8	10.8	10.8	17.2	17.4	17.3	8.9
20190112	Montipora	2990	514	hard	15.7	15.7	15.7	14	14	14	NA

Link to 20191009 TapeStation report, Extractions #495-514

Gel Electrophoresis Results:

20191014 E.S.

DNA/RNA Extractions from Montipora capitata and Pocillopora acuta adult coral fragments from Holobiont Integration Hawaii 2018 project.

Soft and Hard homogenization, and DNA/RNA Extractions followed this protocol. General Zymo Duet DNA/RNA Extraction protocol found here. With the following changes:

• DNA: The first and second elutions were spun down in separate microcentrifuge tubes to compare the quantity and quality between the first and second elution. The first elution had no incubation period (previously 5 minutes) with 10 μl of Tris buffer and the second elution had a 15 minute incubation period (previously 5 minutes) with 100 μl of Tris buffer.
• 10 μl aliquots of DNA and 5 μl aliquots of RNA (instead of 10 μl of each).

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

Timepoint	Species	Coral ID	Extraction ID	Homogenization	DNA Reading 1	DNA Reading 2	Average DNA ng/μl	RNA Reading 1	RNA Reading 2	Average RNA ng/μl	RIN
20181215	Pocillopora	1147	515	soft	113	113	113	147	146	146.5	6.9
20181215	Pocillopora	1147	516	hard	32.4	32.2	32.3	55.2	55.4	55.3	NA
20190112	Montipora	1120	517	soft	25.2	25	25.1	10.6	10.8	10.7	9.1
20190112	Montipora	1120	518	hard	13.1	13.1	13.1	**	**	**	NA

Qubit, Gel, and TapeStation at a later date.

Link to 20191015 TapeStation report, Extractions #25, 177, 179, 279, 477, 493, 515, 517

Gel Electrophoresis Results:

20191015 E.C.

Gel Electrophoresis, Qubit, and TapeStation re-dos for samples that did not read correctly the first time.

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

20191014 Extractions

Timepoint	Species	Coral ID	Extraction ID	Homogenization	DNA Reading 1	DNA Reading 2	Average DNA ng/μl	RNA Reading 1	RNA Reading 2	Average RNA ng/μl	RIN
20181215	Pocillopora	1147	515	soft	113	113	113	147	146	146.5	6.9
20181215	Pocillopora	1147	516	hard	32.4	32.2	32.3	55.2	55.4	55.3	NA
20190112	Montipora	1120	517	soft	25.2	25	25.1	10.6	10.8	10.7	9.1
20190112	Montipora	1120	518	hard	13.1	13.1	13.1	**	**	**	NA

Extraction ID	RNA Reading 1	Reading 2	Average RNA
453	14.2	14.2	14.2
454	10.2	10.2	10.2

TapeStation used to check RNA quality.
Link to 20191015 TapeStation report, Extractions #25, 177, 179, 279, 477, 493, 515, 517

Gel Electrophoresis Results: