Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of the Hawaii TPC samples - 011425

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 trying this time with 25 ng of RNA input, 13 ng and 55.5 ng didn’t work well (see previous posts).

I’ve used the primers in the 96 well plate (CAT. D3096, LOT. 250838) provided with the kit. For sample Mcap-H1, I’ve used UDI primer 4 from the 12 prep kit (CAT. D3008, LOT. 250968). All samples were eluted in 20uL of elution buffer.

Library prep

I ran the library prep protocol linked above. I used 21 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)
Mcap-H1		41.5		0.6		4.4		25
Mcap-G7		17.8		1.4		3.6		25
Pacu-G10		29.9		0.84		4.16		25
Pacu-H9		49.5		0.51		4.49		25
Pacu-F7		34.4		0.73		4.27		25
Mcap-F10		13.8		1.81		3.19		25

I’m re-doing sample Mcap-H1 from the 10th of January. I’m using UDI primer 4 from the 12prep kit (CAT. D3008, LOT. 250968) for this sample. The 96 well plate with the UDI primer is single use, no volume left for re-trying samples.

Qubit Results

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

• Standard 1: 81.16
• Standard 2: 27173.68

QBIT date		sample_id		Species		Temp		DNA read1		DNA read2		DNA_AVG (ng/ul)
20250114		Mcap-H1		Montipora capitata		18		6.32		6.51		6.415
20250114		Mcap-G7		Montipora capitata		35		1.51		1.47		1.49
20250114		Pacu-G10		Pocillopora acuta		26.8		5.38		5.17		5.275
20250114		Pacu-H9		Pocillopora acuta		30		8.33		8.12		8.225
20250114		Pacu-F7		Pocillopora acuta		35		3.95		3.79		3.87
20250114		Mcap-F10		Montipora capitata		26.8		1.15		1.05		1.10

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
20250114		Mcap-H1		Montipora capitata		18		7.93		4
20250114		Mcap-G7		Montipora capitata		35		2.68		26
20250114		Pacu-G10		Pocillopora acuta		26.8		5.73		27
20250114		Pacu-H9		Pocillopora acuta		30		10.0		28
20250114		Pacu-F7		Pocillopora acuta		35		6.37		29
20250114		Mcap-F10		Montipora capitata		26.8		null		30

• Things to consider for next time: 25 ng worked much better than the previous concentrations I tried. I will keep using 25 ng and 21 PCR cycles.