Using Larvae Extraction Protocol on the last 4 of Ariana’s Montipora Coral Recruits on Flash Frozen Plugs

Goal: Use optimized extraction method on 4 plug samples, no homogenization.
Results: Good DNA and RNA quantity, RNA quality is ok, and DNA quality is smeared but similar to previous plug extractions
Take aways: RNA quality and purity was ok, plug_12 had bad purity but it is for RNASeq not TagSeq and might be ok

Sample Info

Note, the whirl packs didn’t have numbers so I gave them IDs

Sample	Timepoint
Plug_9	48hps
Plug_10	48hps
Plug_11	72hps
Plug_12	5dps

Notes

• I did not save any of the plug, I used all of it because there was little material on each one
• I followed the same scrapping protocol as the first plug post post except that I did not do the bead homogenization, only vortexing
• The RNA was eluted in 70ul ultrapure water

Extraction

• Extraction protocol followed exactly
• Extraction proceeded with ~500ul of each sample
• 500ul of DNA/RNA lysis buffer was used for each sample
• 1mL of 100% ethanol was used for each sample
• All samples were eluted for RNA with 40ul ultrapure water in the first elution and 30ul in the second

QC

Qubit

• Broad Range dsDNA and High Sensitivity RNA Qubit protocol
• All samples were read twice
• Qubit was performed and run by Khadija Haider with support from Maggie

BR DNA:

Sample	Standard 1	Standard 2	DNA 1 ng/ul	DNA 2 ng/ul	Average ng/ul
Plug_9	144	15900	51.8	51.2	51.5
Plug_10	-	-	31.6	31.2	31.4
Plug_11	-	-	24	23.4	23.7
Plug_12	-	-	30.8	30.4	30.6

HS RNA:

Sample	Standard 1	Standard 2	RNA 1 ng/ul	RNA 2 ng/ul	Average ng/ul
Plug_9	44	778	75.2	74.8	75
Plug_10	-	-	53.4	53.2	53.3
Plug_11	-	-	31.8	32	31.9
Plug_12	-	-	41.8	41.8	41.8

NanoDrop

• Followed NanoDrop RNA Protocol
• NanoDrop was performed and run by Khadija Haider with support from Maggie

Sample	260/230	260/280
Plug_9	1.44	2.03
Plug_10	1.31	1.95
Plug_11	1.13	1.89
Plug_12	0.22	1.99

Full Results

Traces

Gel

• 1% gel run for 1 hour at 100V
• Gel Protocol
• Gel was made and run by Khadija Haider with support from Maggie

TapeStation

• Followed RNA TapeStation protocol

Sample	RIN score
Plug_9	8
Plug_10	7.8
Plug_11	6.7
Plug_12	6.8

Results Link