Preparing previously sequenced larval samples

I spoke with Katherine last week about combining QuantSeq data from the previous run and the upcoming run. She advised (and I agreed) that it’s optimal for me to re-run those samples. The kits have changed slightly, in addition to me not DNasing the samples previously, her prepping the libraries etc. I have RNA in the -80, so I’m going to catch them up to the other samples today by Turbo DNasing them, and including in the qPCR re-do. Samples are: 34, 35, 37, 39, 41, 44, 45, 47.

DNA contamination re-check with Actin primer set

Today we received a new set of Actin primers that Sam had previously used to check for gDNA contamination. I tested them on one of my larval DNA samples, and voila! My positive control amplified, and the curves appear as expected.

Rather than re-doing ALL samples, I did a sub-set, as I’d like to avoid more thawing/freezing cycles. Oddly, the + control didn’t amplify again with this new primer set.

I discussed the issue with Steven and Sam. Since I did in fact DNase all my samples, I shall move ahead with the RNA concenrating and quality checking.

Quantifying remaining RNA samples after DNasing

In addition to re-running qPCR on 42 samples, I also measured [RNA] for samples that I had not yet quantified due to running out of Qubit tubes. The samples that I did qPCR on were mostly those that also needed quantifying (to minimize freeze/thaw), however I also did some other samples to include a random assortment.

Next step: concentrate dilute samples with Zymo Cleaner/Concentrator

The QuantSeq prep starts with up to 500 ng RNA in 5uL (or less). Katherine advised that I should use no less than 200 or 300 ng. So, here is how much RNA I have in 5 uL:

Tissue	Cohort	Treatment (pCO2, Temp)	RNA Tube #	[RNA] after Dnase treatment (ng/uL)	ng RNA in 0.5 uL	Concentrate?
Ctenidia	Dabob Bay	High	291	158.0	790.0
Ctenidia	Dabob Bay	High	292	29.6	148.0	X
Ctenidia	Dabob Bay	High	293	39.6	198.0	X
Ctenidia	Dabob Bay	High	294	110.0	550.0
Ctenidia	Dabob Bay	High	295	34.8	174.0	X
Ctenidia	Dabob Bay	High	296	180.0	900.0
Ctenidia	Dabob Bay	High	298	182.0	910.0
Ctenidia	Dabob Bay	High	299	50.4	252.0	X
Ctenidia	Dabob Bay	Ambient	301	75.8	379.0
Ctenidia	Dabob Bay	Ambient	302	62.4	312.0
Ctenidia	Dabob Bay	Ambient	306	136.0	680.0
Ctenidia	Dabob Bay	Ambient	304	200.0	1,000.0
Ctenidia	Dabob Bay	Ambient	305	75.2	376.0
Ctenidia	Dabob Bay	Ambient	303	95.2	476.0
Ctenidia	Dabob Bay	Ambient	307	89.4	447.0
Ctenidia	Dabob Bay	Ambient	308	73.6	368.0
Ctenidia	Dabob Bay	Ambient	309	170.0	850.0
Ctenidia	Oyster Bay	High	311	158.0	790.0
Ctenidia	Oyster Bay	High	312	90.6	453.0
Ctenidia	Oyster Bay	High	313	72.4	362.0
Ctenidia	Oyster Bay	High	314	42.2	211.0	X
Ctenidia	Oyster Bay	High	315	148.0	740.0
Ctenidia	Oyster Bay	High	316	146.0	730.0
Ctenidia	Oyster Bay	High	317	158.0	790.0
Ctenidia	Oyster Bay	High	318	174.0	870.0
Ctenidia	Oyster Bay	High	319	77.6	388.0
Ctenidia	Oyster Bay	Ambient	321	148.0	740.0
Ctenidia	Oyster Bay	Ambient	322	44.6	223.0	X
Ctenidia	Oyster Bay	Ambient	323	102.0	510.0
Ctenidia	Oyster Bay	Ambient	324	172.0	860.0
Ctenidia	Oyster Bay	Ambient	325	180.0	900.0
Ctenidia	Oyster Bay	Ambient	326	130.0	650.0
Ctenidia	Oyster Bay	Ambient	327	85.2	426.0
Ctenidia	Oyster Bay	Ambient	328	156.0	780.0
Ctenidia	Oyster Bay	Ambient	329	162.0	810.0
Ctenidia	Fidalgo Bay	High	331	42.2	211.0	X
Ctenidia	Fidalgo Bay	High	332	65.8	329.0
Ctenidia	Fidalgo Bay	High	333	78.6	393.0
Ctenidia	Fidalgo Bay	High	334	64.8	324.0
Ctenidia	Fidalgo Bay	High	335	180.0	900.0
Ctenidia	Fidalgo Bay	High	336	94.8	474.0
Ctenidia	Fidalgo Bay	High	337	194.0	970.0
Ctenidia	Fidalgo Bay	High	338	81.6	408.0
Ctenidia	Fidalgo Bay	High	339	77.2	386.0
Ctenidia	Fidalgo Bay	Ambient	341	89.6	448.0
Ctenidia	Fidalgo Bay	Ambient	342	162.0	810.0
Ctenidia	Fidalgo Bay	Ambient	343	114.0	570.0
Ctenidia	Fidalgo Bay	Ambient	344	25.0	125.0	X
Ctenidia	Fidalgo Bay	Ambient	345	190.0	950.0
Ctenidia	Fidalgo Bay	Ambient	346	43.6	218.0	X
Ctenidia	Fidalgo Bay	Ambient	347	69.0	345.0
Ctenidia	Fidalgo Bay	Ambient	348	54.4	272.0
Ctenidia	Fidalgo Bay	Ambient	349	82.0	410.0
Larvae	Dabob Bay	10 Ambient	401	93.4	467
Larvae	Dabob Bay	10 Ambient	402	114.0	570
Larvae	Dabob Bay	10 Ambient	403	136.0	680
Larvae	Dabob Bay	10 Ambient	404	112.0	560
Larvae	Dabob Bay	10 High	411	72.6	363
Larvae	Dabob Bay	10 High	412	31.2	156	X
Larvae	Dabob Bay	10 High	413	130.0	650
Larvae	Dabob Bay	10 High	414	168.0	840
Larvae	Dabob Bay	6 Ambient	421	57.6	288
Larvae	Dabob Bay	6 High	431b	83.0	415
Larvae	Dabob Bay	6 High	432	74.0	370
Larvae	Dabob Bay	6 High	434	63.0	315
Larvae	Fidalgo Bay	10 Ambient	441	16.2	81	X
Larvae	Fidalgo Bay	10 Ambient	442b	69.8	349
Larvae	Fidalgo Bay	10 Ambient	443	60.2	301
Larvae	Fidalgo Bay	10 Ambient	444	70.6	353
Larvae	Fidalgo Bay	10 Ambient	445	160.0	800
Larvae	Fidalgo Bay	10 High	451	68.4	342
Larvae	Fidalgo Bay	10 High	452b	97.2	486
Larvae	Fidalgo Bay	10 High	453	196.0	980
Larvae	Fidalgo Bay	6 Ambient	461b	84.0	420
Larvae	Fidalgo Bay	6 Ambient	462b	106.0	530
Larvae	Fidalgo Bay	6 High	471b	108.0	540
Larvae	Fidalgo Bay	6 High	472b	97.0	485
Larvae	Fidalgo Bay	6 High	473	124.0	620
Larvae	Fidalgo Bay	6 High	474	77.2	386
Larvae	Fidalgo Bay	6 High	475	27.4	137	X
Larvae	Fidalgo Bay	6 High	476	39.2	196	X
Larvae	Fidalgo Bay	6 High	477	164.0	820
Larvae	Oyster Bay C1	10 Ambient	481	89.2	446
Larvae	Oyster Bay C1	10 Ambient	482	22.2	111	X
Larvae	Oyster Bay C1	10 Ambient	483	99.0	495
Larvae	Oyster Bay C1	10 Ambient	484	58.4	292
Larvae	Oyster Bay C1	10 Ambient	485	19.1	96	X
Larvae	Oyster Bay C1	10 Ambient	486b	148.0	740
Larvae	Oyster Bay C1	10 Ambient	487	118.0	590
Larvae	Oyster Bay C1	10 Ambient	488	60.0	300
Larvae	Oyster Bay C1	10 Ambient	489	68.0	340
Larvae	Oyster Bay C1	10 Ambient	490	186.0	930
Larvae	Oyster Bay C1	10 Ambient	491	58.4	292
Larvae	Oyster Bay C1	10 Ambient	492	82.6	413
Larvae	Oyster Bay C1	10 High	41	108.0	540
Larvae	Oyster Bay C1	10 High	43	156.0	780
Larvae	Oyster Bay C1	10 High	46	98.6	493
Larvae	Oyster Bay C1	10 High	47	128.0	640
Larvae	Oyster Bay C1	10 High	44	41.4	207	X
Larvae	Oyster Bay C1	10 High	506	29.2	146	X
Larvae	Oyster Bay C1	10 High	45	25.1	126	X
Larvae	Oyster Bay C1	6 Ambient	35	94.4	472
Larvae	Oyster Bay C1	6 Ambient	38	LOW	#VALUE!
Larvae	Oyster Bay C1	6 Ambient	513	142.0	710
Larvae	Oyster Bay C1	6 Ambient	39	89.8	449
Larvae	Oyster Bay C1	6 Ambient	37	74.6	373
Larvae	Oyster Bay C1	6 Ambient	34	104.0	520
Larvae	Oyster Bay C1	6 High	521	66.6	333
Larvae	Oyster Bay C1	6 High	522	32.2	161	X
Larvae	Oyster Bay C1	6 High	523	71.4	357
Larvae	Oyster Bay C1	6 High	524	63.0	315
Larvae	Oyster Bay C1	6 High	525	140.0	700
Larvae	Oyster Bay C1	6 High	526	138.0	690
Larvae	Oyster Bay C1	6 High	527	124.0	620
Larvae	Oyster Bay C1	6 High	528	87.6	438
Larvae	Oyster Bay C1	6 High	529	56.8	284
Larvae	Oyster Bay C2	10 Ambient	531	95.4	477
Larvae	Oyster Bay C2	10 Ambient	532	93.6	468
Larvae	Oyster Bay C2	10 Ambient	533	6.5	33	X
Larvae	Oyster Bay C2	10 High	541	44.4	222	X
Larvae	Oyster Bay C2	10 High	542	32.8	164	X
Larvae	Oyster Bay C2	10 High	543	162.0	810
Larvae	Oyster Bay C2	6 Ambient	551	96.4	482
Larvae	Oyster Bay C2	6 Ambient	552b	74.6	373
Larvae	Oyster Bay C2	6 Ambient	553	186.0	930
Larvae	Oyster Bay C2	6 Ambient	554	188.0	940
Larvae	Oyster Bay C2	6 High	561	28.0	140	X
Larvae	Oyster Bay C2	6 High	562	126.0	630
Larvae	Oyster Bay C2	6 High	563	47.0	235	X
Larvae	Oyster Bay C2	6 High	564	152.0	760
Larvae	Oyster Bay C2	6 High	565	31.2	156	X
Control	homog. Control	RNA Control	571	LOW	LOW
Control	homog. Control	RNA Control	572
Control	RNAzol control	RNA Control	574
Control	Turbo Dnase control	RNA Control	575