Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of the Hawaii TPC samples - 012225

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 30 ng of RNA input, 13 ng and 55.5 ng didn’t work well. 25 ng has worked for only some of the samples (see previous posts).

I’ve used the primers in the 96 well plate (CAT. D3096, LOT. 250838) provided with the kit. All samples were eluted in 20uL of elution buffer.

Library prep

I ran the library prep protocol linked above. I used 21 PCR cycles. With 6-8 samples, it takes about 6 hours to run the protocol and do QC on the final library.

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)
Mcap-F8		29.1		1.03		3.97		30
Pcom-G9		62.9		0.48		4.52		30
Pcom-F10		44.0		0.68		4.32		30
Pacu-E8		25.8		1.16		3.84		30
Pacu-H6		36.5		0.82		4.18		30
Pcom-H9		47.1		0.64		4.36		30

Qubit Results

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

• Standard 1: 68.80
• Standard 2: 26748.39

QBIT date		sample_id		Species		Temp		DNA read1		DNA read2		DNA_AVG (ng/ul)
20250122		Mcap-F8		Montipora capitata		25		0.362		0.342		0.352
20250122		Pcom-G9		Porites compressa		30		0.891		0.860		0.8755
20250122		Pcom-F10		Pocillopora acuta		26.8		1.46		1.40		1.43
20250122		Pacu-E8		Pocillopora acuta		25		0.176		0.163		0.1695
20250122		Pacu-H6		Pocillopora acuta		12		0.214		0.195		0.2045
20250122		Pcom-H9		Porites compressa		30		0.371		0.353		0.362

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
20250122		Mcap-F8		Montipora capitata		25		0.577		39
20250122		Pcom-G9		Porites compressa		30		1.36		40
20250122		Pcom-F10		Pocillopora acuta		26.8		2.41		41
20250122		Pacu-E8		Pocillopora acuta		25		0.183		42
20250122		Pacu-H6		Pocillopora acuta		12		0.302		43
20250122		Pcom-H9		Porites compressa		30		0.315		44

• Things to consider for next time: at this point I tried with different amounts of input RNA and most of the samples didn’t work. I will try next time to skip the step in the Zymo protocol that says “for low quality RNA” and see what output I get.