Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of the Hawaii TPC samples - 011525

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 25 ng of RNA input, 13 ng and 55.5 ng didn’t work well (see previous posts).

I’ve used the primers in the 96 well plate (CAT. D3096, LOT. 250838) provided with the kit. All samples were eluted in 20uL 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-H10		54.0		0.42		4.77		25
Pcom-G10		51.8		0.48		4.52		25
Pacu-B1		37.3		0.67		4.33		25
Mcap-B6		12.9		1.94		3.06		25
Mcap-B10		12.1		2.07		2.93		25
Mcap-B1		18.6		1.34		3.66		25
Mcap-E9		22.6		1.11		3.89		25
Pcom-E8		48.1		0.52		4.48		25

Qubit Results

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

• Standard 1: 66.00
• Standard 2: 26812.77

QBIT date		sample_id		Species		Temp		DNA read1		DNA read2		DNA_AVG (ng/ul)
20250115		Pcom-H10		Porites compressa		26.8		2.26		2.26		2.26
20250115		Pcom-G10		Porites compressa		26.8		1.2		1.22		1.21
20250115		Pacu-B1		Pocillopora acuta		18		1.41		1.38		1.395
20250115		Mcap-B6		Montipora capitata		12		1.66		1.62		1.64
20250115		Mcap-B10		Montipora capitata		26.8		1.4		1.35		1.375
20250115		Mcap-B1		Montipora capitata		18		1.45		1.40		1.425
20250115		Mcap-E9		Montipora capitata		30		0.789		0.77		0.7795
20250115		Pcom-E8		Porites compressa		25		1.62		1.55		1.585

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
20250115		Pcom-H10		Porites compressa		26.8		4.07		31
20250115		Pcom-G10		Porites compressa		26.8		1.8		32
20250115		Pacu-B1		Pocillopora acuta		18		1.94		33
20250115		Mcap-B6		Montipora capitata		12		2.87		34
20250115		Mcap-B10		Montipora capitata		26.8		1.91		35
20250115		Mcap-B1		Montipora capitata		18		2.35		36
20250115		Mcap-E9		Montipora capitata		30		1.07		37
20250115		Pcom-E8		Porites compressa		25		3.12		38

• Things to consider for next time: Only one sample, Pcom-H10, seems to have enough concentration for the sequencing (>2 ng/ul). Something during this run of library prep maybe went wrong? Will re-do the other 7 samples.