Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of samples treated for PolyA isolation and pooling - 100125

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.

The first trial of this protocol didn’t work, the number of PCR cycles was probably too low. I’m trying again increasing the number of cycles.

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 21 PCR cycles for the non-NEB treated and 23 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 X2 are NEB-treated, with Y2 non-NEB treated:

sample_id		RNA_QBIT_AVG (ng/uL)		RNA (uL)		Ultrapure water (uL)		Total input RNA (ng)
B8X2		0.574		3.31		1.69		1.9
C10X2		0.71		2.68		2.32		1.9
C9X2		0.393		5		0		1.9
POC_R12_C3X2		0.42		4.52		0.48		1.9
POC_R1_H1X2		0.389		5		0		1.9
POC_R3_C3X2		0.47		4.02		0.98		1.9
B8Y2		21.2		0.94		4.06		20
C10Y2		23.7		0.84		4.16		20
C9Y2		48.9		0.41		4.59		20
POC_R12_C3Y2		28.6		0.7		4.3		20
POC_R1_H1Y2		24.6		0.81		4.19		20
POC_R3_C3Y2		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: 58.50
• Standard 2: 24225.64

QBIT date		sample_id		Species		Library read1		Library read2		Library_AVG (ng/ul)
20251001		B8X2		Montipora capitata		0.356		0.342		0.349
20251001		C10X2		Montipora capitata		1.04		1		1.02
20251001		C9X2		Montipora capitata		0.428		0.42		0.424
20251001		POC_R12_C3X2		Pocillopora acuta		0.738		0.731		0.7345
20251001		POC_R1_H1X2		Pocillopora acuta		0.872		0.869		0.8705
20251001		POC_R3_C3X2		Pocillopora acuta		0.2		0.191		0.1955
20251001		B8Y2		Montipora capitata		0.118		0.111		0.1145
20251001		C10Y2		Montipora capitata		0.104		0.102		0.103
20251001		C9Y2		Montipora capitata		LOW		LOW		none
20251001		POC_R12_C3Y2		Pocillopora acuta		LOW		LOW		none
20251001		POC_R1_H1Y2		Pocillopora acuta		LOW		LOW		none
20251001		POC_R3_C3Y2		Pocillopora acuta		LOW		LOW		none

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
20251001		B8X2		0.249		1
20250929		C10X2		0.756		2
20251001		C9X2		0.554		3
20251001		POC_R12_C3X2		0.768		4
20251001		POC_R1_H1X2		1.06		5
20251001		POC_R3_C3X2		0.076		6
20251001		B8Y2		0.041		9
20250929		C10Y2		/		10
20251001		C9Y2		/		11
20251001		POC_R12_C3Y2		/		12
20251001		POC_R1_H1Y2		0.071		13
20251001		POC_R3_C3Y2		/		14

Libraries pooling

Pooling calculations were done following Zoe’s protocol.

Library_prep_date		Species		Sample.ID		Library_ID		NEB treated or not		Primer		TubeID		Average_Qubit_Conc		TS_Peak_Size		Molarity_nM_Qubit		Amount_Library_0.67nM - POOL 1		Amount_H20_0.67nM - POOL 1		Amount_Library_0.55nM - POOL 2		Amount_H20_0.55nM - POOL 2
20250929		Mcap		B8		B8X		yes		49		B8X		0.54		465		1.76		2.67		4.33
20250929		Mcap		C10		C10X		yes		50		C10X		0.46		522		1.33		3.54		3.46		2.49		3.51
20250929		Mcap		C9		C9X		yes		51		C9X		0.98		525		2.83		1.65		5.35		1.16		4.84
20250929		Mcap		B8		B8Y		no		57		B8Y		0.18		397		0.67		7.00		0.00
20250929		Mcap		C10		C10Y		no		58		C10Y		0.60		349		2.58		1.82		5.18		1.28		4.72
20250929		Mcap		C9		C9Y		no		59		C9Y		0.23		384		0.92		5.08		1.92		3.57		2.43
20250929		Pact		R3		R3_C3Y		no		62		R3Y		0.13		359		0.55						6.00		0.00
20251001		Pact		R3		R3_C3X2		yes		6		R3X2		0.20		538		0.55						6.00		0.00

• I normalized the libraries into 2 pools, one with 0.67nM concentration and one with 0.55nM concentration. Total volume for pool 1 is 7uL*6samples = 42uL, pool 2 is 6uL *6samples = 36uL
• For each library, I added calcucated amount of DNA elution buffer (“Amount_H20”) and the library (“Amount_Library”) to a new PCR tube and mixed well
• I then combined all of these tubes into one LoBind 1.5 mL tube
• I quantified the pooled libraries with Qubit dsDNA HS Assay Kit and tapestation analysis with the D5000 ScreenTape. Then froze until shipping next week

Final pool, sent on 10/06/25 to Oklahoma

Qubit Results

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

• Standard 1: 59.05
• Standard 2: 26461.56

QBIT date		sample_id		Library read1		Library read2		Library_AVG (ng/ul)
20251006		POOL1		0.220		0.202		0.211
20251006		POOL2		0.160		0.152		0.156

TapeStation

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

I only got a peak for POOL1, 468 bp. Next time I’ll run a high sensitivity screentape.