Using the NEBNext Poly(A) Magnetic Isolation Module to isolate mRNA prior in-house library prep

Protocols used

• NEBNext Poly(A) Magnetic Isolation Module
• TapeStation protocol High Sensitivity RNA
• High Sensitivity RNA QBIT protocol

Using the NEBNext Poly(A) Magnetic Isolation Module to isolate intact poly(A)+RNA from previously isolated total RNA. The idea is to use this kit before performing the library prep with the Zymo SwitchFree kit, and try to reduce the amount of rRNA present in the libraries.

The RNA samples I’m using here are from Montipora capitata spat settled on plugs (posts here, here and here).

I followed the kit protocol linked above (express protocol, pages 1-3) and used 200ng of input RNA. The kit is designed to isolate intact poly(A)+ RNA from 1-5µg of previously isolated total RNA.

All samples were eluted in 15uL of elution buffer (NEBNext Tris Buffer).

Sample prep

Here’s a breakdown of input RNA volume and quantity (the kit requires a total starting volume of 50uL):

sample_id		RNA_QBIT_AVG (ng/uL)		RNA (uL)		Ultrapure water (uL)		Total input RNA (ng)
B8		21.2		9.43		40.57		200
C10		23.7		8.44		41.56		200
D1		20.5		9.76		40.24		200
A4		81.5		2.45		47.55		200
A9		40		5.00		45.00		200
D8		46		4.35		45.65		200
C9		48.9		4.09		45.91		200
C13		21.2		9.43		40.57		200
B6		59		3.39		46.61		200
B11		48.2		4.15		45.85		200
D6		60.8		3.29		46.71		200
D11		36.7		5.45		44.55		200
A6		46.4		4.31		45.69		200
C6		27		7.41		42.59		200
D12		30.4		6.58		43.42		200

Qubit Results

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

• Standard 1: 52.910
• Standard 2: 1077.43

QBIT date		sample_id		Species		RNA_read1		RNA_read2		RNA_AVG
20251114		B8		Montipora capitata		0.484		0.483		0.484
20251114		C10		Montipora capitata		0.432		0.43		0.431
20251114		D1		Montipora capitata		0.321		0.319		0.320
20251114		A4		Montipora capitata		0.256		0.254		0.255
20251114		A9		Montipora capitata		0.517		0.511		0.514
20251114		D8		Montipora capitata		0.566		0.561		0.564
20251114		C9		Montipora capitata		0.332		0.331		0.332
20251114		C13		Montipora capitata		0.281		0.269		0.275
20251114		B6		Montipora capitata		0.331		0.329		0.330
20251114		B11		Montipora capitata		0.466		0.46		0.463
20251114		D6		Montipora capitata		0.298		0.297		0.298
20251114		D11		Montipora capitata		0.212		0.21		0.211
20251114		A6		Montipora capitata		0.451		0.449		0.450
20251114		C6		Montipora capitata		0.475		0.472		0.474
20251114		D12		Montipora capitata		0.627		0.621		0.624

TapeStation

After the isolation, I run some of the samples on the tapestation, protocol linked above for high sensitivity. See full report here.

TapeStation date		sample_id		TapeStation conc. ng/uL
20251114		B8		0.590
20251114		C10		0.548
20251114		D1		0.412
20251114		A4		0.567