Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of the Hawaii TPC samples - 012325

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). I will try skipping the step in the Zymo protocol (see above) that is recommended “for low quality RNA”.

I’ve used the primers in 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 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-G9		16.6		1.2		3.8		20
Mcap-H6		21.8		0.92		4.08		20
Mcap-E10		20.9		0.96		4.04		20
Pcom-F6		56.0		0.36		4.64		20
Pacu-G7		39.0		0.51		4.49		20
Pcom-H1		17.3		1.16		3.84		20

Qubit Results

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

• Standard 1: 66.02
• Standard 2: 26117.11

QBIT date		sample_id		Species		Temp		DNA read1		DNA read2		DNA_AVG (ng/ul)
20250123		Mcap-G9		Montipora capitata		30		0.479		0.466		0.4725
20250123		Mcap-H6		Montipora capitata		12		2.45		2.33		2.39
20250123		Mcap-E10		Montipora capitata		26.8		1.25		1.22		1.235
20250123		Pcom-F6		Porites compressa		12		4.11		4.0		4.055
20250123		Pacu-G7		Pocillopora acuta		35		14.2		13.8		14.0
20250123		Pcom-H1		Porites compressa		18		4.95		4.74		4.845

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
20250123		Mcap-G9		Montipora capitata		30		0.491		45
20250123		Mcap-H6		Montipora capitata		12		3.11		46
20250123		Mcap-E10		Montipora capitata		26.8		1.96		47
20250123		Pcom-F6		Porites compressa		12		5.50		48
20250123		Pacu-G7		Pocillopora acuta		35		7.66		49
20250123		Pcom-H1		Porites compressa		18		7.14		50

• Things to consider for next time: skipping the step in the Zymo protocol that says “for low quality RNA” worked much better! I got higher concentrations that previous preps, altough some of the samples have weird peaks (see TapeStation results). I’ll try again skipping that step with samples that didn’t work before and see what happens.