Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of the Hawaii TPC samples - 011125

Protocols used

• Zymo-Seq SwitchFree 3′ mRNA Library Kit protocol
• TapeStation protocol DNA D5000
• High Sensitivity dsDNA QBIT protocol

Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit (CAT. R3009-A, LOT. 252649) to prepare libraries starting from RNA samples exctracted from adult coral fragments from the Hawaii TPC project. RNA extraction posts can be found here.

For the library prep I followed the protocol of Jill Ashey and the kit protocol above. The kit needs a minimum of 10 ng of total RNA or a maximum of 500 ng of total RNA. I’m using the max volume of RNA input (5 ul) for the sample with the lowest RNA concentration among all, which is 11.1 ng/uL, which corresponds to a total of 55.5 ng. I will use this 55.5 as inpout for all other samples.

I’ve used the primers in the 96 well plate (CAT. D3096, LOT. 250838) provided with the kit. For sample Pacu-E9, I’ve used UDI primer 11 from the 12 prep kit (CAT. D3008, LOT. 250968), as I’ve used the day before primer 11 from the 96 plate. All samples were eluted in 20uL of elution buffer.

Library prep

I ran the library prep protocol linked above. I used 20 PCR cycles.

Here’s a breakdown of input RNA volume and quantity:

sample_id		RNA_QBIT_AVG (ng/uL)		RNA (uL)		Ultrapure water (uL)		Total input RNA (ng)
Pacu-E9		64.0		0.87		4.13		55.5
Pacu-H1		20.5		2.71		2.29		55.5
Mcap-G1		53.9		1.03		3.97		55.5
Pcom-E1		18.7		2.97		2.03		55.5
Pcom-B7		22.6		2.46		2.54		55.5
Pacu-H8		27.1		2.05		2.95		55.5
Pacu-G1		60.3		0.92		4.08		55.5
Pcom-E10		27.2		2.04		2.96		55.5

I’m re-doing sample Pacu-E9 from the day before. This time I’m using UDI primer 11 from the 12prep kit (CAT. D3008, LOT. 250968).

Qubit Results

I used High Sensitivity dsDNA Qubit Protocol linked above. DNA samples were read twice, standard only read once.

• Standard 1: 70.26
• Standard 2: 26525.05

QBIT date		sample_id		Species		Temp		DNA read1		DNA read2		DNA_AVG (ng/ul)
20250111		Pacu-E9		Pocillopora acuta		30		1.79		1.75		1.77
20250111		Pacu-H1		Pocillopora acuta		18		1.77		1.79		1.78
20250111		Mcap-G1		Montipora capitata		18		0.708		0.688		0.698
20250111		Pcom-E1		Porites compressa		18		5.00		4.9		4.95
20250111		Pcom-B7		Porites compressa		35		4.48		4.34		4.41
20250111		Pacu-H8		Pocillopora acuta		25		3.31		3.17		3.24
20250111		Pacu-G1		Pocillopora acuta		18		1.11		1.07		1.09
20250111		Pcom-E10		Porites compressa		26.8		3.33		3.29		3.31

TapeStation

After the library prep, I run samples on the tapestation (D5000), protocol linked above.

TapeStation date		sample_id		Species		Temp		TapeStation conc.		Primer set
20250111		Pacu-E9		Pocillopora acuta		30		3.71		11
20250111		Pacu-H1		Pocillopora acuta		18		2.84		12
20250111		Mcap-G1		Montipora capitata		18		1.02		13
20250111		Pcom-E1		Porites compressa		18		5.80		14
20250111		Pcom-B7		Porites compressa		35		4.77		15
20250111		Pacu-H8		Pocillopora acuta		25		4.38		16
20250111		Pacu-G1		Pocillopora acuta		18		1.41		17
20250111		Pcom-E10		Porites compressa		26.8		5.19		18

• Things to consider for next time: Increasing the number of PCR cycles seemed to have imporved the output (better peak profile in TapeStation result), next time I will try with 21 cycles.