Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of the Hawaii TPC samples - 021225

Protocols used

• Zymo OneStep PCR Inhibitor Removal Kit protocol
• 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. The RNA samples I’m using were treated with the Zymo OneStep PCR Inhibitor Removal Kit for removal of potential PCR inhibitors that have caused the library to fail for most of the samples so far.

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).

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 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)
Pcom-G6		35.8		0.56		4.44		20
Pcom-E9		45.9		0.44		4.56		20
Pacu-F9		34.1		0.59		4.41		20
Mcap-F1		17.3		1.16		3.84		20
Pcom-F7		43.4		0.46		4.54		20
Mcap-E7		57.2		0.35		4.65		20
Mcap-F9		16.9		1.18		3.82		20
Pacu-F11		19		1.05		3.95		20

Qubit Results

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

• Standard 1: 67.78
• Standard 2: 26433.03

QBIT date		sample_id		Species		Temp		DNA read1		DNA read2		DNA_AVG (ng/ul)
20250212		Pcom-G6		Porites compressa		12		0.098		0.09		0.094
20250212		Pcom-E9		Porites compressa		30		2.90		2.74		2.82
20250212		Pacu-F9		Pocillopora acuta		30		0.281		0.27		0.276
20250212		Mcap-F1		Montipora capitata		18		0.394		0.362		0.378
20250212		Pcom-F7		Porites compressa		35		0.134		0.120		0.127
20250212		Mcap-E7		Montipora capitata		35		0.654		0.621		0.6375
20250212		Mcap-F9		Montipora capitata		30		1.56		1.49		1.525
20250212		Pacu-F11		Pocillopora acuta		26.8		0.271		0.25		0.2605

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
20250212		Pcom-G6		Porites compressa		12		0		59
20250212		Pcom-E9		Porites compressa		30		4.09		60
20250212		Pacu-F9		Pocillopora acuta		30		0.531		61
20250212		Mcap-F1		Montipora capitata		18		0.686		62
20250212		Pcom-F7		Porites compressa		35		0.0547		63
20250212		Mcap-E7		Montipora capitata		35		1.02		64
20250212		Mcap-F9		Montipora capitata		30		1.31		65
20250212		Pacu-F11		Pocillopora acuta		26.8		0.343		66

• Things to consider for next time: this PCR inhibitor removal kit doesn’t seem to improve things (I’ve tried it on 14 samples and none of them worked), so I will go back to the library prep withou using it.