Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of samples treated for PolyA isolation - 092925

Protocols used

• Zymo-Seq SwitchFree 3′ mRNA Library Kit protocol
• NEBNext Poly(A) Magnetic Isolation Module
• 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 and spat. RNA extraction posts can be found here, here and here, and in Zoe’s notebook (here). NEB treatment post can be found here.

The idea is to use the NEB kit before performing the library prep to reduce the amount of rRNA present in the libraries. Libraries of the same samples non-NEB treated will be also prepared in parallel.

For the library prep I followed the protocol of Jill Ashey and the kit protocol above. I’m using 20 ng of RNA input for the non-NEB treated samples, and 1.9 ng for the NEB-treated samples.

Here I’m using UDI primers from the 96 well plate (CAT. D3096, LOT. 250838) provided with the kit.

All samples were eluted in 15uL of elution buffer.

Library prep

I ran the library prep protocol linked above. I used 19 PCR cycles for the non-NEB treated and 21 for the NEB-treated. 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, samples with X are NEB-treated, with Y non-NEB treated:

sample_id		RNA_QBIT_AVG (ng/uL)		RNA (uL)		Ultrapure water (uL)		Total input RNA (ng)
B8X		0.574		3.31		1.69		1.9
C10X		0.71		2.68		2.32		1.9
C9X		0.393		5		0		1.9
POC_R12_C3X		0.42		4.52		0.48		1.9
POC_R1_H1X		0.389		5		0		1.9
POC_R3_C3X		0.47		4.02		0.98		1.9
B8Y		21.2		0.94		4.06		20
C10Y		23.7		0.84		4.16		20
C9Y		48.9		0.41		4.59		20
POC_R12_C3Y		28.6		0.7		4.3		20
POC_R1_H1Y		24.6		0.81		4.19		20
POC_R3_C3Y		17.7		1.13		3.87		20

Qubit Results

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

• Standard 1: 53.72
• Standard 2: 23477.17

QBIT date		sample_id		Species		Library read1		Library read2		Library_AVG (ng/ul)
20250929		B8X		Montipora capitata		0.54		0.538		0.539
20250929		C10X		Montipora capitata		0.458		0.456		0.457
20250929		C9X		Montipora capitata		0.992		0.972		0.982
20250929		POC_R12_C3X		Pocillopora acuta		0.16		0.16		0.16
20250929		POC_R1_H1X		Pocillopora acuta		0.232		0.228		0.23
20250929		POC_R3_C3X		Pocillopora acuta		LOW		LOW		LOW
20250929		B8Y		Montipora capitata		0.176		0.174		0.175
20250929		C10Y		Montipora capitata		0.594		0.596		0.595
20250929		C9Y		Montipora capitata		0.238		0.23		0.234
20250929		POC_R12_C3Y		Pocillopora acuta		0.132		0.132		0.132
20250929		POC_R1_H1Y		Pocillopora acuta		0.104		0.11		0.107
20250929		POC_R3_C3Y		Pocillopora acuta		0.134		0.128		0.131

TapeStation

After the library prep, I run samples on the tapestation (D5000), protocol linked above. Full tapestation report here

TapeStation date		sample_id		TapeStation conc. ng/uL		Primer set
20250929		B8X		0.056		49
20250929		C10X		0.74		50
20250929		C9X		0.05		51
20250929		POC_R12_C3X		/		52
20250929		POC_R1_H1X		/		53
20250929		POC_R3_C3X		0.06		54
20250929		B8Y		0.46		57
20250929		C10Y		0.68		58
20250929		C9Y		1.14		59
20250929		POC_R12_C3Y		0.03		60
20250929		POC_R1_H1Y		0.04		61
20250929		POC_R3_C3Y		0.02		62