Trying again with library prep using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for Bermuda TPC samples - Mcav and Dlab

Protocols used

• Zymo-Seq SwitchFree 3′ mRNA Library Kit protocol
• Zymo Clean & Concentrator-5 kit protocol
• QBIT protocol
• TapeStation protocol HS RNA
• TapeStation protocol DNA D5000

Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit to try again preparing libraries starting from RNA samples exctracted from adult coral fragments from the Hawaii and Bermuda TPC project. The library prep for Mcav and Dlab RNA samples has not worked so far, so I’m trying again by first cleaning the RNA with the Zymo Clean & Concentrator-5 kit and slightly increasing the input RNA concentration from 13 to 15ng. Cleaning the RNA should get rid of potential PCR inhibitors that might cause failure of the library prep for these species.

For the library prep I followed the protocol of Jill Ashey and the kit protocol linked above. The kit needs a minimum of 10 ng of total RNA or a maximum of 500 ng of total RNA. I’m using 15 ng and the same number of cycles as my previous attempts, 21 PCR amplification cycles (maximum recommended by the zymo kit for <100ng input RNA).

RNA clean up

I’ve followed the Zymo Clean & Concentrator-5 kit protocol above and this post by Zoe. The minimun sample volume for this kit is 50ul and the column binding capacity is 10ng of RNA, so I’m taking an aliquot from the original RNA sample and adding RNAse-free water. I will do the cleanup on just the Mcav-F1 sample, then do the library prep for the cleaned Mcav-F1, non-cleaned Mcav-F1 and an additional non-cleaned Dlab-A1 sample.

Here’s a breakdown of input RNA volume for the RNA clean-up (QBIT and extraction by Zoe, see here):

sample_id		RNA_QBIT_AVG (ng/uL)		RNA (uL)		Ultrapure water (uL)		Total starting volume (ul)
Mcav-F1		34.4		10		40		50

Sample eluted after the cleanup in 8ul.

QBIT after RNA cleanup

I measured the RNA concentration of the cleaned Mcav-F1 RNA, of the original Mcav-F1 RNA sample (non-cleaned), and of the non-cleaned Dlab-A1 sample. I used the high sensitivity assay.

• Mcav-F1 cleaned: 7.82 ng/ul read 1, 7.44 read 2
• Mcav-F1 non-cleaned: 15.0 ng/ul read 1, 13.9 read 2 *much lower than Zoe’s initial qbit
• Dlab-A1 non-cleaned: 12.6 ng/ul read 1, 12.0 read 2

TapeStation after RNA cleanup

I run the Mcav-F1 cleaned and non-cleaned on the TS using the High Sensitivity reagents and tape, protocol linked above. The RIN of the cleaned RNA sample is lower than the non-cleaned but still ok for the library prep kit (>4).

Tape station result for Dlab-A1

Library prep

I run the library prep protocol linked above, with the 2 Mcav-F1 cleaned and non-cleaned samples and Dlab-A1 sample (extracted by Zoe, see here). Input RNA 15 ug total.

Here’s a breakdown of input RNA volume:

sample_id	RNA_QBIT_AVG (ng/uL)	RNA (uL)	Ultrapure water (uL)	Total starting volume (ul)
Mcav-F1_clean	7.63	1.97	3.03	5.0
Mcav-F1	14.45	1.04	3.96	5.0
Dlab-A1	12.6	1.19	3.81	5.0

TapeStation after library prep

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

Library prep date		sample_id		Species		Temp		TapeStation conc.		Primer set
20241126		Mcav-F1_clean		Montastraea cavernosa		29		Below 1		8
20241126		Mcav-F1		Montastraea cavernosa		29		Below 1		9
20241126		Dlab-A1		Diploria labyrinthiformis		29		Below 1		10