Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of the Hawaii TPC samples - 012425

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 20 ng of RNA input, 13 ng, 30 ng and 55.5 ng didn’t work well. 25 ng has worked for only some of the samples (see previous posts). Skipping the step in the Zymo protocol that says “for low quality RNA” worked with the last batch of samples (see post of 23rd), so I will try again with these samples skipping it.

Here I’m using primers 39-44 that we already had in the lab, and that were diluted and mixed (5i+i7). The primers in the 96 well plate (CAT. D3096, LOT. 250838) provided with the kit are single use, so could not reuse them again. All samples were eluted in 18uL 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. I’ve re-prepped the following samples, that had failed on the 22nd, skipping the “low quality RNA” step according to the Zymo kit protocol above.

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		0.69		4.31		20
Pcom-G9		62.9		0.32		4.68		20
Pcom-F10		44.0		0.45		4.55		20
Pacu-E8		25.8		0.78		4.22		20
Pacu-H6		36.5		0.55		4.45		20
Pcom-H9		47.1		0.42		4.58		20

Qubit Results

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

• Standard 1: 66.42
• Standard 2: 27992.47

QBIT date		sample_id		Species		Temp		DNA read1		DNA read2		DNA_AVG (ng/ul)
20250124		Mcap-F8		Montipora capitata		25		0.057		0.051		0.054
20250124		Pcom-G9		Porites compressa		30		LOW		LOW
20250124		Pcom-F10		Pocillopora acuta		26.8		LOW		LOW
20250124		Pacu-E8		Pocillopora acuta		25		LOW		LOW
20250124		Pacu-H6		Pocillopora acuta		12		0.072		0.07		0.071
20250124		Pcom-H9		Porites compressa		30		LOW		LOW

*LOW: out of range

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
20250124		Mcap-F8		Montipora capitata		25		0		39
20250124		Pcom-G9		Porites compressa		30		0		40
20250124		Pcom-F10		Montipora capitata		26.8		0		41
20250124		Pacu-E8		Porites compressa		12		0		42
20250124		Pacu-H6		Pocillopora acuta		35		0		43
20250124		Pcom-H9		Porites compressa		18		0.054		44

• Things to consider for next time: I got nothing for all samples on the Tapestation, which is strange. I think it might have been because of the primers that I used (not part of the kit, already in the lab)