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Sample inventory In general, samples that were transported at room temperature were stored in the -20ºC freezer, while those transported in the dry shipper were either left in the dry shipper or stored at -70ºC. Those highlighted in yellow are at NMMBA (Taiwan) for long-term storage. Those highlighted in purple are in -20ºC freezer at 842 N. Capital Ave., Indy (USA) Those highlighted in green were sent to Nikki Traylor-Knowles in October, 2016. Location 1 Location 2 buffer notes Australs-Cooks, April-May 2013 (extractions were performed at ULL in Oct.-Dec., 2013 and Jan., 2014) N=122, of which ~90 were analyzed fully (gene expression [NMMBA] and genotyping [NMMBA and ULL, 88 in 1st Platax manuscript]) 3 boxes in Indiana, 1.5 boxes of DNA @ NMMBA, 1 box of tissues to send to Nikki Tissues (molecular)a -20ºC (1 box @ NMMBA; n=25) RNALater All homogenized 92 with Nikki (homogenized in RNALater) Tissues (microscopy)b NMMBA office n=28 RNA (concentrated) -20ºC (1 box @ Indy) Several @ NMMBA DEPC-H20 No cDNAs remain (used all) RNA (diluted) -20ºC (1 box @ Indy) Sufficient for one additional RT rxn 20 ng/ul DNA (concentrated) -20ºC (1 box) @ NMMBA Axygen eluent No diluted DNAs remain(w/Hollie) protein -20ºC 1 box @ Indy) 1.4 ml acetone Purify at later date Fiji, June 2013 (extractions were performed at ULL in March-April, 2014) N=153, of which 90 were analyzed for gene expression, and 96 have been genotyped as of 9-7-16. Tissues (molecular)c -20ºC (2 boxes @ NMMBA, 80-90 remain) RNALater All but one were homogenized 73 in RNALater (all homogenized) with Nikki Tissues (microscopy)b NMMBA office n=33 RNA (concentrated) -20ºC (1.5 boxes @ Indy) DEPC-H20 RNA (diluted) -20ºC (1.5 boxes @ Indy) DEPC-H20 20 ng/ul DNA (concentrated) -20ºC (3 boxes) @ NMMBA Axygen eluent No cDNAs remain (used all) DNA (diluted) -20ºC (3 boxes) @ Axygen eluent + 10 ng/ul NMMBA DEPC-H20 protein -20ºC (1 box) @ Indy -20ºC (1 box) @ NMMBA 1.4 ml acetone Purify at later date Tonga, September 2013 (extractions were performed at ULL in Fall-Winter 2014-2015) N=115, of which 90 were analyzed (45 from Ha’apai and 45 from Va’vau) for gene expression/genotyping Tissues (molecular)c,d -20ºC (2 boxes @ Give to Nikki or Luisa in RNALater, plant Extracted @ ULL in Oct. Dec., NMMBA) early 2017 lysis buffer 2014 and Jan. 2015 Tissues (microscopy)b NMMBA office n=31 RNA (concentrated) -20ºC (2-3 boxes) DEPC-H20 Concentrated and diluted are in n=90 same box DNA (concentrated) -20ºC (2 boxes) @ Axygen eluent Concentrated and diluted are in n=90 NMMBA same box DNA (diluted) -20ºC (2 boxes) @ Axygen eluent+ No cDNAs remain NMMBA milliQ water Protein (precipitated) -20ºC (1 box) Acetone or Purify at later date isopropanol New Caledonia, November 2013 (perform extractions in mid-2015 at NMMBA) N=139, 120 analyzed in September, 2015. Tissues (molecular)c -20ºC (2 boxes) -20ºC (1 box, transferred RNALater Extracted @ NMMBA in July 2015 Only process nonto -70C in Feb. 2015) (homogenized and homogenized samples non-homogenized RNA (concentrated, -20ºC NMMBA (2 boxes) DEPC-H20 (30 ul) n=120) RNA (diluted, n=120) -20ºC NMMBA (2 boxes) DEPC-H20 (21 ul) DNA (concentrated, -20ºC NMMBA (2 boxes) “Eluent” (50 ul) n=120) DNA (diluted, n=120) -20ºC NMMBA (2 boxes) DEPC-H20 (50 ul) cDNA (5-10) -20ºC NMMBA (1 box) DEPC-H20 (80 ul) Used all for most samples Protein (n=130-140) -20ºC NMMBA (1 main box, rest over several boxes) Precipitated in acetone Tissues (microscopy)b NMMBA office paraffin n=28+6 w/ missing labels Solomon Islands, November 2014 (performed extractions in 2016) all samples except for several diluted DNAs are at NMMBA Pocillopora sp. (mostly P. acuta) and N=200 Seriatopora sp. (mix of SH and SC, gave to Noodle in early Feb. 2015) Tissues (molecular) -20ºC freezer in RBII Transported all samples to Taiwan N=126 in Feb. 2015 Tissues (microscopy) n=36 NMMBA office paraffin RNA (concentrated, -20ºC freezer in RBII n=120) (across 2 boxes) RNA (diluted, n=120) -20ºC freezer in RBII (across 2 boxes) DNA (concentrated, -20ºC freezer in RBII (across two boxes) n=138) DNA (diluted, n=100) Indy (10-20 samples) Mailed remainder ~(100) Used all cDNA for assays to Nikki in Sept. 2016 (Solaris+8 genes) Protein (n=130-140) -20ºC freezer in RBII (across two boxes) Palau, January 2015, 185 samples across 150 unique colonies, five night dives, may have been at room temperature for 2-3 days Tissues (molecular, n=185) -20ºC freezer in RBII RNALater (none were homogenized) TRIzol (n=113) Only process TRIzol samples Tissues (microscopy, n=35) NMMBA office PBS Embedded by BB RNA (concentrated, n=160) 2 boxes in -20ºC freezer DEPC-H20 (30 ul) in RBII RNA (diluted, n=160) 2 boxes in -20ºC freezer DEPC-H20 (11 ul) in RBII cDNA (n=150) Indy DNA (concentrated, n=160) 2 boxes -20ºC freezer in Protech kit eluent RBII (50 ul) DNA (diluted, n=160) Indy DEPC-H20 (50 ul) Protein (n=160) -20ºC freezer in RBII acetone (~1.5 ml) Chagos, March-April 2015 (two missions): 166 P. acuta/damicornis samples (140 in TRIzol to process), 174 Seriatopora spp. samples, and 312 other Pocillopora sp. samples for Joao Tissues (molecular) 2 boxes (n=166) NMMBA RNALater (1.5-1.7 Took to Taiwan in May 2015 at RT -80C ml/sample) Tissues (molecular) 2 boxes (n=140) Gave to Pi in on 9-20-16 TRIzol (650-1,500 Took to Taiwan in May 2015 at RT 2016 (-20C) ul/sample) 1.5 ml PBS, EDTA, or FSW Seriatopora spp. 3 boxes (n=175) 1.5 ml DESS Seriatopora spp. 1 box (n=30) in -20C @ Gave to Pi in October 1.5 ml TRIzol NMMBA 2016 1.7 ml RNALater Joao samples 3 boxes (n=312, Taken by Andy 1.5 ml DESS or 156/cruise) 80% ethanol Maldives, January, 2016 (two weeks): 87 Pocillopora spp. and 79 Acropora spp. samples Tissues (molecular) 0.5 box -20ºC @ NMMBA RNALater First ~30 samples only Give to Luisa in Oct. 2016 Tissues (molecular) 2 boxes Gave to Pi on 9-20-16 TRIzol All 166 samples (n=166) Tissues (molecular) 2 boxes (post-bleaching) -20ºC @ Indy RNALater N=? Dongsha, March, 2016: 28 P. acuta/damicornis samples in TRIzol only 1 box in Pi’s -20ºC a All samples were transported from ship to ULL at room temperature. b Samples were fixed, washed, decalcified, washed w/ PBS, & transported from the ship to ULL (& later to Taiwan) in PBS. c Some samples were transported from the ship to ULL via dry shipper (-150ºC). d Some samples are stored in GeneMark plant lysis buffer (non-homogenized). Tissues (microscopy) NMMBA office Australs-Cooks +1 box with concentrated RNA +1 box with diluted RNAs (both concentrated and diluted) +1 box with proteins precipitated in acetone (some samples in other boxes) =3 total in Indy freezer + 1.5 boxes of RNA and DNA (concentrated only) and 1 box of remaining tissues @ NMMBA Fiji 1.5 boxes with RNA (concentrated and diluted; some RNAs are in Taiwan) 1 box with proteins precipitated in acetone = 2.5 total in Indy+ 6.5 boxes @ NMMBA (see list below) Tonga: +2 boxes with RNA (concentrated and diluted) +1 box with proteins = 2.5 total in Indy + 4 boxes of DNA @ NMMBA Solomon Islands 10-20 DNAs in box with Maldives samples. Palau 2 boxes with cDNAs +2 boxes with diluted DNAs +concentrated and diluted RNAs, precipitated proteins, concentrated DNAs, and all tissues are at NMMBA Additional boxes at Indy -20C freezer 1. primer box 1 includes DNase and some other reagents 2. primer box 2 Total boxes in Indiana: 3 A/C boxes (including remaining reagents)+ 2.5 Fiji boxes+2.5 Tonga boxes+2 Maldives boxes (post-bleaching)+2 primers boxes =12 boxes in freezer posi tion 1 2 3 4 NMMBA samples. Green=pack up and bring to US to give to Nikki. -20C rack 1: reagents and NSF -20C rack 2: NSF rack 2 and LOF rack -20C rack 3: LOF rack 2: samples 1: A/C and Fiji Fiji, Tonga, and NC PCR reagents & primers NO SLOT NO SLOT PCR reagents (Hsiao) Misc. NSF DNAs and cDNA NO SLOT PCR/cloning reagents (Hsiao) Misc. NSF samples Fiji DNA Lab reagents PDLTTE DNA, cDNA, and TEM Fiji DNA (diluted) -20C rack 4: LOF rack 4: New Caledonia and Dongsha NO SLOT NC tissues NC RNAs (concentrated) 1-62 NC RNAs (concentrated) 63-104 5 SHVTS primers 6 Symbiodinium primers 7 PD and other primers 8 Cardboard primer box (mine and Hsiao’s) 9 Hsiao’s primers 10 PDpCO2 DNAs 11 SHVTS tissues and DNAs capsules PDSTTE#2 cDNA, DNA, and Pi’s mesocosm samples Misc. NSF samples and Pi’s pCO2 mesocosm samples: tissues in TRIzol Pi’s pCO2 mesocosm samples: tissues in TRIzol Australs/Cooks DNAs Fiji DNAs and tissues -20C rack 5: LOF rack 5: Solomon Islands 1 2 3 4 5 6 7 8 9 10 Solomon Islands tissues in RNALater+proteins Solomon Islands tissues in RNALater Solomon Islands tissues in RNALater and PLB Solomon Islands RNAs Solomon Islands RNAs (diluted) Solomon Islands diluted RNAs and cDNAs Solomon Islands DNAs Solomon Islands DNAs (diluted) Solomon Islands DNAs (diluted) Solomon Islands proteins Fiji DNA (diluted) w/ some Australs-Cooks DNA (~10 samples) Fiji: proteins and tissues and proteins NC RNAs (diluted) 1-75 Tonga tissues NC diluted RNAs, DNA, and cDNAs (n=10 only) NC DNAs (concentrated) 1-76 Tonga tissues + DNAs NC DNAs (concentrated) 77-104D Tonga DNAs 1-49 (concentrated & diluted) Tonga DNAs 50-98 (concentrated & diluted) N NC tissues in RNALater+ some proteins -20C rack 6: Solomon Islands, Palau, Chagos, and Maldives Solomon Islands RNAs and proteins Palau tissues (TRIzol and RNALater) #1 Palau tissues (TRIzol and RNALater) #2 Palau RNAs #1 Palau RNAs #2 Palau DNAs #1 Palau DNAs #2 Palau proteins #1 Palau proteins #2 Palau mixed NC DNAs diluted: 1-81 NC proteins Dongsha samples: 1-28 -20C rack 7: Palau cDNA Chagos 1-PD in RNALater and TRIzol Chagos 2-PD in TRIzol Chagos 3-Seriatopora in TRIzol Ruth’s samples in guanidinium Maldives tissues #1 Maldives tissues #2 HTT primers and reagents Rack #8 in office (can hold 10 more boxes). Will generate 8-10 boxes for 166 Chagos samples Maldives tissues #3 LOF sample processing: Green = completed. Yellow=in progress. Red=2016. Cruise (#) Dates Sample Extractions GCPb Symbiodinium Host Gene expression a size genotype genotype Australs/ Apr.-May 123/28 100/123 60/10 60/100 89/100 Solarisd+6 Sym gene Cooks (1) 2013 0 0 host genesc Fiji (2) June 2013 153/33 91/153 90/91 90/91 94/153 Solaris+ 4 Sym genes+ 4 host genes Tonga (3) Sept. 2013 115/31 90/115 90/90 90/90 ~112/115 Solaris+4 Sym genes+ 4 host genes New (4) Nov. 2013 139/28 120/139 120/ 120/120 ~100 Solaris+4 Sym genes+ Caledonia 125 4 host genes GBR (5) Sept. 2014 No sampling Solomon Oct.-Nov. 125/35 138/145 120/ (n=120) 111 Solaris+4 Sym Islands (6) 2014 120 genes+4 host genes Palau (7) Jan. 2015 185/35 150/185 150/ 150 n=150/150 Fall 2016 Solaris+4 Sym genes + 4 host genes (target Notes/other/to-do 1. Image analysis 1. Write manuscript 1. Morphology differences 2. Species difference 1. Light-dark differences within a sample 1. Analyze data 1. Light-dark differences within a sample 2. Healthy-bleached within sample differences 1. Light-dark differences within a sample new genes) Chagos (8-9) Chagos (8-9) Maldivese (10) Dongshae (11) TOTAL Mar.-May 2015 Mar.-May 2015 166/70 Poc. Sp. Extract from 150 (TRIzol only) 30 Seriatopora sp. in TRIzol. Extract all winter 2017 winter 2017 winter 2017 winter 2017 2. Healthy-bleached within sample differences 1. Naturally bleached transcriptome/proteome 1. healthy vs. bleached proteomics 2. Noodle has 174 samples 1. Pre-, during and postbleaching comparison 2. Pre- and post-bleaching 87 Pocillopora spp. Spring 2017 Spring 2017 79 Acropora spp. Mar. 2016 28 Pocillopora Late 2017 Late 2017 damicornis/acuta ~1,000 Pocillopora sp. (1,034) 620 dives 1. ~575 Seriatopora sp. (574-600) aThe first and second values represent the total for molecular and histological analyses, respectively. bproxy for Symbiodinium density. cdid not assess due to having mix of different species. dexogenous control enot LOF samples. Jan. 2016 LOF samples. Those in yellow are in the US (Indianapolis), while those in purple are in Taiwan. Samples highlighted in green are with Nikki in Miami. The RNAs are either fully concentrated (typically around 100 ng/ul) or diluted to 20 ng/ul. The DNAs are either fully concentrated (typically around 150 ng/ul) or diluted to 10 ng/ul. The cDNAs are all the equivalent of 200 ng RNA (i.e., 200 ng RNA was used in the RT rxn). Location Tissues (buffer) Tissues (form) RNA cDNA DNA protein Paraffin TEM/SEM blocks Typically TRIzol® or frozen in Every form imaginable ✔ Taiwan ✔ ✔ ✔ ✔ ✔ FSW (n=~150 over various (nubbins, slurry, small buffers) projects) fragments, etc.) RNALater (n=61) Homogenized only ✔✔a ✔b ✔ (precipitated ✔ Austral Islands ) a RNALater Homogenized only ✔✔ ✔ (precipitated ✔ Cook Islands ✔ ✔ (n=61) ) (precipitated ✔ (precipitated ✔ (precipitated ✔ (precipitated ✔ (precipitated ✔ 5) Late 2016 ✔ 60) 2017 0 Fiji RNALater (n=153) Homogenized only ✔ ✔ ✔ Tonga RNALater (n=115) ✔ ✔ ✔ New Caledonia RNALater (n=150) ✔ ✔ ✔ Solomon Islandsc RNALater (n=125) Homogenized & unhomogenized Homogenized & unhomogenized unhomogenized ✔ ✔ ✔✔✔ Palau RNALater (n=150) TRIzol (n=113) RNALater (n=165) TRIzol (n=165) RNALater (n=30) TRIzol (n=166) RNALater (n=?) TRIzol only (n=28) unhomogenized ✔ ✔ ✔ unhomogenized unhomogenized Chagos (i.e., BIOT) Maldives (not LOF samples) ✔ ) ✔ ) ✔ ) ✔ ) ✔ ) Dongsha (not LOF unhomogenized Not suitable for skeleton analysis 0 samples) Total ~1,200 (though many, if not the majority, are NOT the alpha genotype (i.e., the archetypal P. damicornis) a Would need to re-synthesize cDNA. bSome diluted DNAs are Hollie. cAlso have Seriatopora hystrix from this cruise onwards (~100 colonies/cruise).