Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of the Hawaii TPC samples - 011325

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 13 ng of input RNA, since it’s the amount the gave me the highest concentration of library with the first samples I tried prepping. Note: Using more input RNA (55.5 ng) got me low library concentration (<2n ng/ul) for many samples so far. Maybe issue with contaminants inhibiting PCR?

I’ve used the primers in the 96 well plate (CAT. D3096, LOT. 250838) provided with the kit. For sample Pacu-H1, I’ve used UDI primer 12 from the 12 prep kit (CAT. D3008, LOT. 250968), as I’ve used the day before primer 12 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 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)
Pacu-H1		20.5		0.64		4.37		13
Pacu-F8		17.9		0.73		4.27		13
Pcom-F8		14.4		0.90		4.10		13
Mcap-G6		21.2		0.61		4.39		13
Mcap-H9		27.6		0.47		4.53		13
Pcom-H6		16.2		0.80		4.20		13
Pcom-B6		21.2		0.61		4.39		13
Mcap-B9		18.7		0.70		4.30		13

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. Library samples were read twice, standard only read once.

• Standard 1: 70.54
• Standard 2: 27629.65

QBIT date		sample_id		Species		Temp		DNA read1		DNA read2		DNA_AVG (ng/ul)
20250113		Pacu-H1		Pocillopora acuta		18		0.476		0.376		0.426
20250113		Pacu-F8		Pocillopora acuta		25		1.06		0.91		0.985
20250113		Pcom-F8		Porites compressa		25		3.31		2.99		3.15
20250113		Mcap-G6		Montipora capitata		12		2.61		2.31		2.46
20250113		Mcap-H9		Montipora capitata		30		1.31		1.18		1.245
20250113		Pcom-H6		Porites compressa		12		1.68		1.57		1.625
20250113		Pcom-B6		Porites compressa		12		LOW		LOW		LOW
20250113		Mcap-B9		Montipora capitata		30		0.299		0.301		0.3

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
20250113		Pacu-H1		Pocillopora acuta		18		0.746		12
20250113		Pacu-F8		Pocillopora acuta		25		1.84		19
20250113		Pcom-F8		Porites compressa		25		5.02		20
20250113		Mcap-G6		Montipora capitata		12		4.55		21
20250113		Mcap-H9		Montipora capitata		30		2.48		22
20250113		Pcom-H6		Porites compressa		12		2.88		23
20250113		Pcom-B6		Porites compressa		12		0.034		24
20250113		Mcap-B9		Montipora capitata		30		0.6		25

• Things to consider for next time: Decreasing the input RNA didn’t help, samples look worse. I will increase the input again to 25ng, and keep 21 PCR cycles.