Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of the Hawaii TPC samples after removal of PCR inhibitors - 020525

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. 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. See the post here about PCR inhibitors removal.

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-G10		13.0		1.54		3.46		20
Pcom-F8		17.8		1.12		3.88		20
Pacu-H1		11.5		1.74		3.26		20
Pacu-D8		13.3		1.57		3.43		20
Mcap-G1		12.7		1.60		3.40		20
Mcap-F10		12.5		1.50		3.50		20

Qubit Results

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

• Standard 1: 75.28
• Standard 2: 28194.13

QBIT date		sample_id		Species		Temp		DNA read1		DNA read2		DNA_AVG (ng/ul)
20250205		Pcom-G10		Porites compressa		26.8		0.222		0.210		0.216
20250205		Pcom-F8		Porites compressa		25		too low		too low		too low
20250205		Pacu-H1		Pocillopora acuta		18		0.699		0.701		0.700
20250205		Pacu-D8		Pocillopora acuta		25		too low		too low		too low
20250205		Mcap-G1		Montipora capitata		18		0.432		0.430		0.431
20250205		Mcap-F10		Montipora capitata		26.8		0.439		0.426		0.4325

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
20250205		Pcom-G10		Porites compressa		26.8		0.208		20
20250205		Pcom-F8		Porites compressa		25				32
20250205		Pacu-H1		Pocillopora acuta		18		1.41		12
20250205		Pacu-D8		Pocillopora acuta		25				13
20250205		Mcap-G1		Montipora capitata		18		0.508		30
20250205		Mcap-F10		Montipora capitata		26.8		0.389		57