Throubleshooting library prep using the Zymo-Seq SwitchFree 3′ mRNA Library Kit following recommendations from Zymo team - Hawaii TPC samples - 051325

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 this library prep (performed on 05/11/25.), I followed the protocol of Jill Ashey and the kit protocol above. I used the Zymo team recommendations to troubleshoot the library prep as detailed here. In particular, I’ve done the following:

1. I’ve used 10 ng as input RNA.
2. I’ve decreased the volume of Poly A R1 Reagent from 5 ul to 3 ul (supplementing with 2 ul of DNase/RNase Free water)
3. I’ve used 19 PCR cycles, final elution 20 ul.
4. I’ve performed an extra cleanup (following Appendix C of the Zymo kit manual above) on individual libraries (I didn’t pool them). With the extra cleanup, I eluted in 15 ul instead of 20 ul.
5. I’ve let the beads to dry for 6-7 minutes.

Here I’m using UDI primers for WGBS that we had in the lab.

Library prep

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)
Pacu-F9		34.1		0.29		4.71		10
Pcom-F7		43.4		0.23		4.77		10
Mcap-F1		17.3		0.58		4.42		10

The library prep for these samples didn’t work before, see previous post.

Qubit Results

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

• Standard 1: 82.18
• Standard 2: 27184.51

QBIT date		sample_id		Species		Library read1		Library read2		Library_AVG (ng/ul)
20250511		Pacu-F9		Pocillopora acuta		19.1		18.7		18.9
20250511		Pcom-F7		Porites compressa		4.9		4.7		4.8
20250511		Mcap-F1		Montipora capitata		6.5		6.1		6.3

TapeStation

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

TapeStation date		sample_id		Species		TapeStation conc.		Primer set
20250513		Pacu-F9		Pocillopora acuta		23.2		34
20250513		Pcom-F7		Porites compressa		5.88		35
20250513		Mcap-F1		Montipora capitata		7.46		36