Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of the Hawaii TPC samples - 021325

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 try again using 25 ng of RNA input, 13 ng, 30 ng and 55.5 ng didn’t work well. 20 ng has worked for only some of the samples (see previous posts).

Here I’m using UDI primers from the 96 well plate (CAT. D3096, LOT. 250838) provided with the kit. All samples were eluted in 18uL of elution buffer.

Library prep

I ran the library prep protocol linked above. I’m trying with 22 PCR cycles (so far I used the max recommended by the kit, which is 21). 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-G11		14		1.79		3.21		25
Pcom-F9		46.8		0.53		4.47		25
Pacu-B10		153.5		0.16		4.84		25
Pacu-G6		43.8		0.57		4.43		25
Mcap-H7		42.9		0.58		4.42		25
Mcap-E1		48.7		0.51		4.49		25
Pacu-G9		13.9		1.80		3.20		25
Pcom-E6		24.3		1.03		3.97		25

Qubit Results

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

• Standard 1: 66.40
• Standard 2: 28121.89

QBIT date		sample_id		Species		Temp		DNA read1		DNA read2		DNA_AVG (ng/ul)
20250213		Mcap-G11		Montipora capitata		26.8		0.826		0.786		0.806
20250213		Pcom-F9		Porites compressa		30		0.289		0.278		0.284
20250213		Pacu-B10		Pocillopora acuta		26.8		1.52		1.43		1.475
20250213		Pacu-G6		Pocillopora acuta		12		0.784		0.699		0.742
20250213		Mcap-H7		Montipora capitata		35		2.68		2.58		2.63
20250213		Mcap-E1		Montipora capitata		18		5.10		4.84		4.97
20250213		Pacu-G9		Pocillopora acuta		30		2.3		2.25		2.275
20250213		Pcom-E6		Porites compressa		12		0.403		0.380		0.392

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
20250213		Mcap-G11		Montipora capitata		26.8		1.63		67
20250213		Pcom-F9		Porites compressa		30		0.198		68
20250213		Pacu-B10		Pocillopora acuta		26.8		2.41		69
20250213		Pacu-G6		Pocillopora acuta		12		1.26		70
20250213		Mcap-H7		Montipora capitata		35		4.48		71
20250213		Mcap-E1		Montipora capitata		18		5.67		72
20250213		Pacu-G9		Pocillopora acuta		30		5.96		73
20250213		Pcom-E6		Porites compressa		12		0.599		74

• Things to consider for next time: increasing the number of PCR cycles to 22 doesn’t improve things. Need to re-think about a strategy with these samples.