July 12, 2019

Homogenized the rest of the Oly ctenidia tissues, as described in the previous post. For most samples I acheived at minimum 30 mg tissue. The scale was not very sensitive, so I’m estimating an error of +/- 20mg.

Tested the RNAzol isolation process on 6 samples, one from each cohort x treatment. Followed the RNAzol® RT RNA Isolation Reagent protocol for Total RNA isolation, using half of my homogenate, so 0500 uL.

RNAzol Steps:

• Labeled 1.7 mL tubes with sample numbers, 2 tubes per sample
• Added 200 uL DEPC-treated water to one set of tubes
• Transferred 500 uL of homogenate to tubes with water, returned rest of homogenate (500 uL) to -20 freezer.
• Vortexed homogenate + water vigorously 15 seconds
• Held mixture for 15 minutes at room temperature
• Centrifuged at 12,000 g (aka rcf) for 15 minutes. NOTE: for this first batch of 6 samples, I accidentally centrifuged at 1,200 for 15 minutes, and didn’t catch the error until later.
• Transfered supernatant to new tubes (used 2nd set of labeled tubes). DNA/proteins/polysaccharides remain in the bottom of the tubes. I added the label “DNA + Pr.” to these tubes, and saved in the -20 freezer.
• Added 0.75 mL isopropanol to precipitate RNA
• Vortexed vigorously for 15 seconds. Held for 10 minutes at room temperature
• Centrifuged at 12,000 rcf for 10 minutes
• Discarded supernatant. A white RNA pellet was visible in samples (which is good)
• Added 500 uL 75% ethanol, which I prepared using the 200 proof ethanol and DEPC-treated water.
• Mixed by hand, made sure the pellete was in the ethanol, then centrifuged at 8,000 rcf for 3 minutes.
• Discarded ethanol supernatant, repeated previous step, then discarded ethanol again.
• Added 50 uL DEPC-treated water, vortexted by hand for 10 seconds.
• Stored RNA dissolved in water in the -20 freezer for now.

July 15, 2019

Another batch of RNA isolation today. I ran 24 samples at once, which I won’t do again, as it takes too long per step. Otherwise, things went well and there were no changes to the above protocol. One note: sample 295 had no visible RNA pellet, so I don’t expect I’ll find any RNA when I quantify. This was probably due to the small size of the initial tissue piece, and loss during mortar/pestle homogenization.

July 16, 2019

Finished isolating ctenidia RNA today. Ran 2 batches of 12 samples, which allowed me to finish more quickly, and ultimately didn’t take much longer tha doing 24 in one batch. Good to know for the future. Again, no changes to protocol, but some notes:

• The last batch accidentally sat for 25 minutes during the initial homogenate + water step (should have been 15 minutes) - timer malfunction :/
• Dissolved RNA pellet in 150 uL DEPC treated water, not 50 uL. See notes below.

RNA Quantification, round 1

Quantifed 18 samples, 3 from each treatment x cohort. Used 5 uL (of 50 uL), and the Qubit RNA assay kit (high sensitivity). All except 1 sample (#302) read “TOO HIGH”. I chatted with Sam, and based on the size of my RNA pellets he suggested dissolving RNA in 150 uL water, not 50 uL, and using 1 uL with quantifying.

Based on the above, I added 100 uL of DEPC-treated water to all samples that I had previously processed. NOTE: this means that the 18 samples that I already quantified now have a total volume of ~145 uL, while the rest will have 150 uL, with a few exceptions:

• I did not add more water to sample 302, as it quantified in the first round.
• Samples 295, X and Y: only added 50 uL water, as no pellets were visible.

RNA Quantification, round 2

Quantified all ctenidia samples (except for #302) using Qubit HS RNA assay. Prepared the 53 samples, plus the two standards. Thawed samples, then transferred 1 uL into Qubit working solution (prepared using 60 uL reagent + 11,940 uL buffer). Good news is that I have enough RNA in all samples - see table in repo, also pasted below - need to dilute a few samples and re-quantify before proceeding to the QuantSeq library prep.

Cohort	pCO2	HOMOGENATE TUBE #	TUBE COLOR	TISSUE TYPE	TISSUE SAMPLE #	VOL RNAzol	MASS TISSUE (mg)	DATE HOMOGENIZED	Homogenization Batch	DATE RNA ISOLATED	RNA Isolation Batch	Total volume, uL (RNA + H2O)	[RNA] ng/uL	Amount of RNA (ng)	Volume needed for 500 ng RNA	Notes
Dabob Bay	High	291	PURPLE	CTENIDIA	HL10-10	1 mL	80-100	7/10/19	1	7/12/19	1	144	200	28,800	2.50
Dabob Bay	High	292	PURPLE	CTENIDIA	HL10-11	1 mL	10-30	7/10/19	1	7/15/19	2	144	47.8	6,883	10.46
Dabob Bay	High	293	PURPLE	CTENIDIA	HL10-12	1 mL	10-30	7/10/19	1	7/15/19	2	144	47.2	6,797	10.59
Dabob Bay	High	294	PURPLE	CTENIDIA	HL6-10	1 mL	80-100	7/10/19	4	7/15/19	2	149	108	16,092	4.63
Dabob Bay	High	295	PURPLE	CTENIDIA	HL6-11	1 mL	10-30	7/10/19	4	7/15/19	2	49	49.6	2,430	10.08
Dabob Bay	High	296	PURPLE	CTENIDIA	HL6-12	1 mL	60-80	7/12/19	5	7/16/19	3	149	198	29,502	2.53
Dabob Bay	High	297	PURPLE	CTENIDIA	HL6-13	1 mL	< 10	7/12/19	5	7/16/19	3	149	4.2	626	119.05
Dabob Bay	High	298	PURPLE	CTENIDIA	HL6-14	1 mL	80-100	7/12/19	7	7/16/19	4	149	76.4	11,384	6.54	Black substance in homogenate - maybe from tube lid?
Dabob Bay	High	299	PURPLE	CTENIDIA	HL6-15	1 mL	10-30	7/12/19	7	7/16/19	4	149	56.8	8,463	8.80
Dabob Bay	Ambient	301	RED	CTENIDIA	HL10-19	1 mL	10-30	7/10/19	1	7/12/19	1	144	82.6	11,894	6.05
Dabob Bay	Ambient	302	RED	CTENIDIA	HL10-20	1 mL	10-30	7/10/19	1	7/15/19	2	45	36.8	1,656	13.59
Dabob Bay	Ambient	306	RED	CTENIDIA	HL10-21	1 mL	50-70	7/10/19	2	7/15/19	3	144	200	28,800	2.50
Dabob Bay	Ambient	304	RED	CTENIDIA	HL6-19	1 mL	50-70	7/10/19	1	7/15/19	2	149	TOO HIGH	>28,000	#VALUE!
Dabob Bay	Ambient	305	RED	CTENIDIA	HL6-20	1 mL	20-40	7/10/19	1	7/15/19	2	149	82	12,218	6.10
Dabob Bay	Ambient	303	RED	CTENIDIA	HL6-21	1 mL	80-100	7/12/19	1	7/16/19	2	149	120	17,880	4.17
Dabob Bay	Ambient	307	RED	CTENIDIA	HL6-16	1 mL	70-100	7/12/19	4	7/16/19	3	149	95.4	14,215	5.24
Dabob Bay	Ambient	308	RED	CTENIDIA	HL6-17	1 mL	70-90	7/12/19	4	7/16/19	4	149	63.6	9,476	7.86
Dabob Bay	Ambient	309	RED	CTENIDIA	HL6-18	1 mL	80-100	7/12/19	6	7/16/19	4	149	200	29,800	2.50
Oyster Bay	High	311	ORANGE	CTENIDIA	SN6-16	1 mL	80-100	7/10/19	2	7/12/19	1	144	174	25,056	2.87
Oyster Bay	High	312	ORANGE	CTENIDIA	SN6-17	1 mL	80-100	7/10/19	2	7/15/19	2	144	97.4	14,026	5.13
Oyster Bay	High	313	ORANGE	CTENIDIA	SN6-18	1 mL	Not recorded	7/10/19	3	7/15/19	2	144	76.2	10,973	6.56
Oyster Bay	High	314	ORANGE	CTENIDIA	SN6-19	1 mL	Not recorded	7/10/19	3	7/15/19	2	149	49.6	7,390	10.08
Oyster Bay	High	315	ORANGE	CTENIDIA	SN6-20	1 mL	70-100	7/10/19	4	7/15/19	2	149	TOO HIGH	>28,000	#VALUE!
Oyster Bay	High	316	ORANGE	CTENIDIA	SN6-21	1 mL	100 +	7/12/19	4	7/16/19	3	149	TOO HIGH	>28,000	#VALUE!
Oyster Bay	High	317	ORANGE	CTENIDIA	SN6-22	1 mL	70-90	7/12/19	5	7/16/19	3	149	TOO HIGH	>28,000	#VALUE!
Oyster Bay	High	318	ORANGE	CTENIDIA	SN6-23	1 mL	60-80	7/12/19	6	7/16/19	4	149	182	27,118	2.75
Oyster Bay	High	319	ORANGE	CTENIDIA	SN6-24	1 mL	20-40	7/12/19	7	7/16/19	4	149	61.8	9,208	8.09
Oyster Bay	Ambient	321	YELLOW	CTENIDIA	SN6-25	1 mL	60-80	7/10/19	2	7/12/19	1	144	188	27,072	2.66
Oyster Bay	Ambient	322	YELLOW	CTENIDIA	SN6-26	1 mL	20-40	7/10/19	2	7/15/19	2	144	63.4	9,130	7.89
Oyster Bay	Ambient	323	YELLOW	CTENIDIA	SN6-27	1 mL	20-40	7/10/19	3	7/15/19	2	144	110	15,840	4.55
Oyster Bay	Ambient	324	YELLOW	CTENIDIA	SN6-28	1 mL	40-60	7/10/19	3	7/15/19	2	149	164	24,436	3.05
Oyster Bay	Ambient	325	YELLOW	CTENIDIA	SN6-29	1 mL	70-100	7/10/19	5	7/15/19	2	149	200	29,800	2.50
Oyster Bay	Ambient	326	YELLOW	CTENIDIA	SN6-30	1 mL	70-100	7/12/19	5	7/16/19	3	149	168	25,032	2.98
Oyster Bay	Ambient	327	YELLOW	CTENIDIA	SN6-31	1 mL	40-60	7/12/19	6	7/16/19	3	149	94.6	14,095	5.29
Oyster Bay	Ambient	328	YELLOW	CTENIDIA	SN6-32	1 mL	50-70	7/12/19	6	7/16/19	4	149	176	26,224	2.84
Oyster Bay	Ambient	329	YELLOW	CTENIDIA	SN6-33	1 mL	70-100	7/12/19	7	7/16/19	4	149	150	22,350	3.33
Fidalgo Bay	High	331	GREEN	CTENIDIA	NF6-16	1 mL	40-60	7/10/19	2	7/12/19	1	144	56.4	8,122	8.87
Fidalgo Bay	High	332	GREEN	CTENIDIA	NF6-17	1 mL	10-40	7/10/19	2	7/15/19	2	144	63.4	9,130	7.89
Fidalgo Bay	High	333	GREEN	CTENIDIA	NF6-18	1 mL	40-60	7/10/19	3	7/15/19	2	144	82.8	11,923	6.04
Fidalgo Bay	High	334	GREEN	CTENIDIA	NF6-19	1 mL	30-50	7/10/19	3	7/15/19	2	149	66.2	9,864	7.55
Fidalgo Bay	High	335	GREEN	CTENIDIA	NF6-20	1 mL	90-110	7/10/19	4	7/15/19	2	149	TOO HIGH	>28,000	#VALUE!
Fidalgo Bay	High	336	GREEN	CTENIDIA	NF6-21	1 mL	90-110	7/12/19	4	7/16/19	3	149	130	19,370	3.85
Fidalgo Bay	High	337	GREEN	CTENIDIA	NF6-22	1 mL	100 +	7/12/19	6	7/16/19	3	149	198	29,502	2.53
Fidalgo Bay	High	338	GREEN	CTENIDIA	NF6-23	1 mL	20-40	7/12/19	6	7/16/19	4	149	83.8	12,486	5.97
Fidalgo Bay	High	339	GREEN	CTENIDIA	NF6-24	1 mL	10-30	7/12/19	7	7/16/19	4	149	78.6	11,711	6.36
Fidalgo Bay	Ambient	341	BLUE	CTENIDIA	NF6-25	1 mL	20-50	7/10/19	2	7/12/19	1	144	92.4	13,306	5.41
Fidalgo Bay	Ambient	342	BLUE	CTENIDIA	NF6-26	1 mL	80-100	7/10/19	3	7/15/19	2	144	178	25,632	2.81
Fidalgo Bay	Ambient	343	BLUE	CTENIDIA	NF6-27	1 mL	50-70	7/10/19	3	7/15/19	2	144	146	21,024	3.42
Fidalgo Bay	Ambient	344	BLUE	CTENIDIA	NF6-28	1 mL	40-70	7/10/19	5	7/15/19	2	149	28.4	4,232	17.61
Fidalgo Bay	Ambient	345	BLUE	CTENIDIA	NF6-29	1 mL	70-100	7/10/19	5	7/15/19	2	149	TOO HIGH	>28,000	#VALUE!
Fidalgo Bay	Ambient	346	BLUE	CTENIDIA	NF6-30	1 mL	20-40	7/12/19	5	7/16/19	3	149	74	11,026	6.76
Fidalgo Bay	Ambient	347	BLUE	CTENIDIA	NF6-31	1 mL	10-30	7/12/19	6	7/16/19	3	149	89.8	13,380	5.57
Fidalgo Bay	Ambient	348	BLUE	CTENIDIA	NF6-32	1 mL	50-70	7/12/19	6	7/16/19	4	149	51.4	7,659	9.73
Fidalgo Bay	Ambient	349	BLUE	CTENIDIA	NF6-33	1 mL	10-40	7/12/19	7	7/16/19	4	149	55.4	8,255	9.03