diff --git a/assays/ClockGating/isa.assay.xlsx b/assays/ClockGating/isa.assay.xlsx index 00f281fada198983d567158a72c06c75aded2ea8..5f94cdcb4348bf9b25661cb415bc1346c86a9428 100644 Binary files a/assays/ClockGating/isa.assay.xlsx and b/assays/ClockGating/isa.assay.xlsx differ diff --git a/assays/ClockGating/protocols/RNAExtractionCDNASynthesis.txt b/assays/ClockGating/protocols/RNAExtractionCDNASynthesis.txt index 9fb0cc8c176d40377da2446c5d74169c04159262..ff10c472089cd8940621885314aabe1cab3ef9c3 100644 --- a/assays/ClockGating/protocols/RNAExtractionCDNASynthesis.txt +++ b/assays/ClockGating/protocols/RNAExtractionCDNASynthesis.txt @@ -1,3 +1,2 @@ - Total RNA was isolated from whole seedlings using the NucleoSpin RNA Plant Kit (Macherey-Nagel). First-strand cDNA was synthesized using the PrimeScript RT Reagent Kit (Perfect Real Time, Takara Bio). Reverse transcription-mediated quantitative real-time PCR (RT–qPCR) analyses were performed on an AriaMx Real-Time PCR System (Agilent) using Absolute Blue Low Rox Mix (Thermo Fisher Scientific). PROTEIN 19 PHOSPHATASE2a subunit A3 (PP2A, AT1G13320) and TIP41 (AT4G34270) were used as reference genes to calculate relative expression values (2ΔCt values). Primer sequences have been described previously (Anwer et al., 2020). The primers used for PRR7 were forward: 5ʹ-TGAAAGTTGGAAAAGGACCA-3ʹ and reverse:5ʹ-GTTCCACGTGCATTAGCTCT-3ʹ. \ No newline at end of file diff --git a/assays/IRImaging16C22C/isa.assay.xlsx b/assays/IRImaging16C22C/isa.assay.xlsx index e1c8c19d6cfa75641eeaee1a171c804620622f6e..6df6bb45e7a32405e847fea3b2eb842553d710e1 100644 Binary files a/assays/IRImaging16C22C/isa.assay.xlsx and b/assays/IRImaging16C22C/isa.assay.xlsx differ diff --git a/assays/IRImaging16C22C/protocols/IRImaging16C22C.txt b/assays/IRImaging16C22C/protocols/IRImaging16C22C.txt new file mode 100644 index 0000000000000000000000000000000000000000..742af92b8fc55cd4d112806080bf6706bc353c74 --- /dev/null +++ b/assays/IRImaging16C22C/protocols/IRImaging16C22C.txt @@ -0,0 +1,3 @@ +Infrared-based growth rate and cotyledon movement analysis (IR Imaging): + +To allow unobstructed visualization of hypocotyl and cotyledons in air, seedlings were grown vertically on the agar ledge formed by removing part of the agar in the square plate as previously described (Anwer et al., 2020). To image growth in day-night cycles we built an infrared imaging platform consisting of a modified camera with IR long pass 830 nm cut filters (Panasonic G5). Illumination was achieved using 880 nm IR backlights (Kingbright BL0106-15-29). Imaging was started at Zeitgeber time (ZT) 00 on day 3 (after germination). Photographs were taken every 60 min for 96 h in constant light (LL, white fluorescent lamps: 30 μmol m–2 s–1) under specified thermocycles (12 h 22 °C:12 h 16 °C or 12 h 28 °C:12 h 22 °C). The imaging platform with infrared illumination was previously described (Anwer et al., 2020). Image stacks were analyzed using ImageJ (http://imagej.nih.gov/ij/). The circadian parameters of cotyledon movement were determined using the MFourFit method integrated in the BioDare2 analysis platform (Zielinski et al., 2014). diff --git a/assays/IRImagingClockMutants/isa.assay.xlsx b/assays/IRImagingClockMutants/isa.assay.xlsx index a5a357eb33470ad444bc26bdfdf6993ea1966604..166cafcdf143474310d9ec6121c05f4cdbc3ed56 100644 Binary files a/assays/IRImagingClockMutants/isa.assay.xlsx and b/assays/IRImagingClockMutants/isa.assay.xlsx differ diff --git a/assays/IRImagingClockMutants/protocols/IRImagingCockMutants.txt b/assays/IRImagingClockMutants/protocols/IRImagingCockMutants.txt new file mode 100644 index 0000000000000000000000000000000000000000..742af92b8fc55cd4d112806080bf6706bc353c74 --- /dev/null +++ b/assays/IRImagingClockMutants/protocols/IRImagingCockMutants.txt @@ -0,0 +1,3 @@ +Infrared-based growth rate and cotyledon movement analysis (IR Imaging): + +To allow unobstructed visualization of hypocotyl and cotyledons in air, seedlings were grown vertically on the agar ledge formed by removing part of the agar in the square plate as previously described (Anwer et al., 2020). To image growth in day-night cycles we built an infrared imaging platform consisting of a modified camera with IR long pass 830 nm cut filters (Panasonic G5). Illumination was achieved using 880 nm IR backlights (Kingbright BL0106-15-29). Imaging was started at Zeitgeber time (ZT) 00 on day 3 (after germination). Photographs were taken every 60 min for 96 h in constant light (LL, white fluorescent lamps: 30 μmol m–2 s–1) under specified thermocycles (12 h 22 °C:12 h 16 °C or 12 h 28 °C:12 h 22 °C). The imaging platform with infrared illumination was previously described (Anwer et al., 2020). Image stacks were analyzed using ImageJ (http://imagej.nih.gov/ij/). The circadian parameters of cotyledon movement were determined using the MFourFit method integrated in the BioDare2 analysis platform (Zielinski et al., 2014). diff --git a/assays/WhiteLightImaging16C22CLD/dataset/ZZH-20-014_LD_rawdata_Fig1A.csv b/assays/WhiteLightImaging16C22CLD/dataset/ZZH-20-014_LD_rawdata_Fig1A.csv index 1b80703f86884d7682f380183cfa0521cc64732c..2e5997943ed99ac27125eee613462cb6c737fae8 100644 --- a/assays/WhiteLightImaging16C22CLD/dataset/ZZH-20-014_LD_rawdata_Fig1A.csv +++ b/assays/WhiteLightImaging16C22CLD/dataset/ZZH-20-014_LD_rawdata_Fig1A.csv @@ -1,3 +1,3 @@ version https://git-lfs.github.com/spec/v1 -oid sha256:6ff26152cae05cad9e1608c41e7beac32f1a193babba8a280fef067c5ee4c29a -size 10955 +oid sha256:f3befbcd8c1bd00fda7fad64182e9b87b220685d108d861c6923139399a8acd1 +size 14914 diff --git a/assays/WhiteLightImaging16C22CLD/isa.assay.xlsx b/assays/WhiteLightImaging16C22CLD/isa.assay.xlsx index 8325c5630b5a42bc4d2e21f93d0c97c135589eff..78442e6baf69132fa79ff1da2ba47ec41d33dc80 100644 Binary files a/assays/WhiteLightImaging16C22CLD/isa.assay.xlsx and b/assays/WhiteLightImaging16C22CLD/isa.assay.xlsx differ diff --git a/assays/WhiteLightImaging16C22CLD/protocols/WhiteLightImagingLD.txt b/assays/WhiteLightImaging16C22CLD/protocols/WhiteLightImagingLD.txt new file mode 100644 index 0000000000000000000000000000000000000000..38b6390c386beb83ec49c22f99811e651a4eaaaa --- /dev/null +++ b/assays/WhiteLightImaging16C22CLD/protocols/WhiteLightImagingLD.txt @@ -0,0 +1,5 @@ +White Light Imaging LD: + +8 days old seedlings were imaged using a Nikon D60 DSLR camera and hypocotyl length was measured using ImageJ (http://imagej.nih.gov/ij/). + + diff --git a/assays/WhiteLightImaging16C22CSD/dataset/ZZH-20-014_SD_rawdata_Fig1B.csv b/assays/WhiteLightImaging16C22CSD/dataset/ZZH-20-014_SD_rawdata_Fig1B.csv index 75a6d696456cc76d6ba12dd030efb4bfc015ebdb..8264a275e42dd407ac3afcee3724c83bd72370f8 100644 --- a/assays/WhiteLightImaging16C22CSD/dataset/ZZH-20-014_SD_rawdata_Fig1B.csv +++ b/assays/WhiteLightImaging16C22CSD/dataset/ZZH-20-014_SD_rawdata_Fig1B.csv @@ -1,3 +1,3 @@ version https://git-lfs.github.com/spec/v1 -oid sha256:5282715f48d10402f958b1e794fb79ca9ee6cd71d7ec4185ef3e203f197f1f21 -size 8954 +oid sha256:de09ba48f0aa5a951adf545ec216a090807bf143207d9c4bec3e63832991fc49 +size 12164 diff --git a/assays/WhiteLightImaging16C22CSD/isa.assay.xlsx b/assays/WhiteLightImaging16C22CSD/isa.assay.xlsx index 3fa8fee351c928ffcaff8284d5653824a2f4a8c1..6170ee600e680bf328a815679ad16d61b5239262 100644 Binary files a/assays/WhiteLightImaging16C22CSD/isa.assay.xlsx and b/assays/WhiteLightImaging16C22CSD/isa.assay.xlsx differ diff --git a/assays/WhiteLightImaging16C22CSD/protocols/WhiteLightImagingSD.txt b/assays/WhiteLightImaging16C22CSD/protocols/WhiteLightImagingSD.txt new file mode 100644 index 0000000000000000000000000000000000000000..08eb079b31ade60d1ed1268451d03b6fd4325498 --- /dev/null +++ b/assays/WhiteLightImaging16C22CSD/protocols/WhiteLightImagingSD.txt @@ -0,0 +1,5 @@ +White Light Imaging SD: + +8 days old seedlings were imaged using a Nikon D60 DSLR camera and hypocotyl length was measured using ImageJ (http://imagej.nih.gov/ij/). + + diff --git a/assays/qRTexpressionTimecourse/isa.assay.xlsx b/assays/qRTexpressionTimecourse/isa.assay.xlsx index 0c5b7a8143b058a7cd1eea3a7e08cafcd4e3e478..1dddf536f5eb7f6a9207b9f519c93cfa899ccb45 100644 Binary files a/assays/qRTexpressionTimecourse/isa.assay.xlsx and b/assays/qRTexpressionTimecourse/isa.assay.xlsx differ diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx index f866ceaf715f1821a0335f61b6a54d26ce06431b..6b3930b357d6b68a10f2e3143a358d11f3a46508 100644 Binary files a/isa.investigation.xlsx and b/isa.investigation.xlsx differ diff --git a/studies/ELF3TempEntrainment/isa.study.xlsx b/studies/ELF3TempEntrainment/isa.study.xlsx index 581cb0a9a6f3639708c527442732fea901ebde2f..0e12d5a8f8bf505ae100730b9d53aa24e4490461 100644 Binary files a/studies/ELF3TempEntrainment/isa.study.xlsx and b/studies/ELF3TempEntrainment/isa.study.xlsx differ diff --git a/studies/ELF3TempEntrainment/protocols/ClockGatingExpressionAnalysis.txt b/studies/ELF3TempEntrainment/protocols/ClockGatingExpressionAnalysis.txt new file mode 100644 index 0000000000000000000000000000000000000000..46fe1f2113e22d8082131d195c2f10b0d6b7e743 --- /dev/null +++ b/studies/ELF3TempEntrainment/protocols/ClockGatingExpressionAnalysis.txt @@ -0,0 +1,3 @@ +Clock Gating Expression Analysis: + +For the temperature gating assay, seedlings were entrained in constant light (LL, 90 μmol m–2 s–1), under 12 h 22 °C:12 h 16 °C thermocycles for 8 d. On day 9, starting from ZT00, seedlings were either treated with a 4 h temperature pulse (28 °C) at various ZTs, or were kept under the same conditions (no treatment) before samples were harvested at the specified time. All experiments were performed using three biological replicates. diff --git a/studies/ELF3TempEntrainment/protocols/IRImaging16C22C.txt b/studies/ELF3TempEntrainment/protocols/IRImaging16C22C.txt new file mode 100644 index 0000000000000000000000000000000000000000..40ad35fc062c633a6d6400b57d2f86b73b9f04ce --- /dev/null +++ b/studies/ELF3TempEntrainment/protocols/IRImaging16C22C.txt @@ -0,0 +1,3 @@ +Infrared-based growth rate and cotyledon movement analysis (IR Imaging): + +To allow unobstructed visualization of hypocotyl and cotyledons in air, seedlings were grown vertically on the agar ledge formed by removing part of the agar in the square plate as previously described (Anwer et al., 2020). To image growth in day-night cycles we built an infrared imaging platform consisting of a modified camera with IR long pass 830 nm cut filters (Panasonic G5). Illumination was achieved using 880 nm IR backlights (Kingbright BL0106-15-29). Imaging was started at Zeitgeber time (ZT) 00 on day 3 (after germination). Photographs were taken every 60 min for 96 h in constant light (LL, white fluorescent lamps: 30 μmol m–2 s–1) under specified thermocycles (12 h 22 °C:12 h 16 °C or 12 h 28 °C:12 h 22 °C). diff --git a/studies/ELF3TempEntrainment/protocols/IRImagingCockMutants.txt b/studies/ELF3TempEntrainment/protocols/IRImagingCockMutants.txt new file mode 100644 index 0000000000000000000000000000000000000000..d03bd3fa74073635d65220084ab94e62679109dd --- /dev/null +++ b/studies/ELF3TempEntrainment/protocols/IRImagingCockMutants.txt @@ -0,0 +1,3 @@ +Infrared-based growth rate and cotyledon movement analysis (IR Imaging): + +To allow unobstructed visualization of hypocotyl and cotyledons in air, seedlings were grown vertically on the agar ledge formed by removing part of the agar in the square plate as previously described (Anwer et al., 2020). To image growth in day-night cycles we built an infrared imaging platform consisting of a modified camera with IR long pass 830 nm cut filters (Panasonic G5). Illumination was achieved using 880 nm IR backlights (Kingbright BL0106-15-29). Imaging was started at Zeitgeber time (ZT) 00 on day 3 (after germination). Photographs were taken every 60 min for 96 h in constant light (LL, white fluorescent lamps: 30 μmol m–2 s–1) under specified thermocycles (12 h 22 °C:12 h 16 °C or 12 h 28 °C:12 h 22 °C). diff --git a/studies/ELF3TempEntrainment/protocols/PlantMaterialGrowthConditions.txt b/studies/ELF3TempEntrainment/protocols/PlantMaterialGrowthConditions.txt deleted file mode 100644 index 0747dc41162942957f0a47fd3290caa96066d3c0..0000000000000000000000000000000000000000 --- a/studies/ELF3TempEntrainment/protocols/PlantMaterialGrowthConditions.txt +++ /dev/null @@ -1,21 +0,0 @@ -Plant Material: - -All A. thaliana lines used were in the Ws-2 or the Columbia-0 (Col-0) background. The elf3-4 (Zagotta et al., 1996; Hicks et al., 2001), gi-158 and elf3-4 gi-158 (Anwer et al., 2020), phyB-10 (Feldmann, 1991; Franklin et al., 2003), and pcl1-2 (Onai and Ishiura, 2005) null mutants in the Ws-2 background have been described previously. The elf3-4 phyB-10 was generating by crossing. The elf4-2, elf4-2 ELF3-OE, and elf3-1 ELF4-OE mutants in the Col-0 background have likewise been described previously (Nusinow et al., 2011; Box et al., 2015; Jung et al., 2020). Ws-2, elf3-4, phyB-10, and elf3-4 phyB-10 additionally harbor a CCR2:LUC reporter construct, and Ws-2 and pcl1-2 additonally harbor GI:LUC: - -White Light Imaging: - -Unless stated otherwise, seedlings were grown on vertically oriented plates in long days (LDs, 16 h light:8 h dark) or short days (SDs, 8 h light:16 h dark) with 90 μmol m–2 s-1 photosynthetically active radiation (PAR) using white fluorescent lamps (T5 4000K). Seedlings were grown at constant 16 °C or 22 °C for 8 d, or at constant 20 °C or 28 °C for 8 d. For temperature shift assays, seedlings grown at 20 °C for 4 d were shifted to 28 °C or were kept at 20 °C for an additional 4 d. For assays in constant light (LL, 90 -μmol m–2 s–1), seedlings were grown at constant 16, 22, or 28 °C for 8 d. Seedlings were imaged, and hypocotyl length was measured using ImageJ (http://imagej.nih.gov/ij/). Temperature response (%) was calculated by the fold change of each measured hypocotyl length at higher temperature (22 °C or 28 °C) relative to the median hypocotyl length at lower temperature (16 °C or 20 °C). - -Infrared-based growth rate and cotyledon movement analysis (IR Imaging): - -To allow unobstructed visualization of hypocotyl and cotyledons in air, seedlings were grown vertically on the agar ledge formed by removing part of the agar in the square plate as previously described (Anwer et al., 2020). Imaging was started at Zeitgeber time (ZT) 00 on day 3 (after germination). Photographs were taken every 60 min for 96 h in constant light (LL, white fluorescent lamps: 30 μmol m–2 s–1) under specified thermocycles (12 h 22 °C:12 h 16 °C or 12 h 28 °C:12 h 22 °C). For free-running conditions, seedlings were entrained by thermocycles for 2 d after germination in LL and then on day 3 at ZT00 were released into constant conditions (30 μmol m–2 s–1 light and 22 °C temperature). The imaging platform with infrared illumination was previously described (Anwer et al., 2020). Image stacks were analyzed using ImageJ (http://imagej.nih.gov/ij/). The circadian parameters of cotyledon movement were determined using the MFourFit method integrated in the BioDare2 analysis platform (Zielinski et al., 2014). The relative amplitude error (RAE) analysis was used to estimate the robustness of the circadian rhythm: RAE values range from 0 to 1, where 0 represents a robust -rhythm, and 1 represents no rhythm. - -qRT Timecourse expression Analysis: - -Seedlings were entrained in constant light (LL, 90 μmol m–2 s–1) or darkness (DD), under 12 h 22 °C:12 h 16 °C thermocycles for 8 d. On day 9, starting from ZT00, the samples were harvested every 4 h. - -Clock Gating Expression Analysis: - -For the temperature gating assay, seedlings were entrained under thermocycles (with LL) as described above for 8 d. On day 9, starting from ZT00, seedlings were either treated with a 4 h temperature pulse (28 °C) at various ZTs, or were kept under the same conditions (no treatment) before samples were harvested at the specified time. All experiments were performed using three biological replicates. diff --git a/studies/ELF3TempEntrainment/protocols/TimeCourseExpressionAnalysis.txt b/studies/ELF3TempEntrainment/protocols/TimeCourseExpressionAnalysis.txt new file mode 100644 index 0000000000000000000000000000000000000000..4fa994a5cde2de195c566e8953eeb3f441698a23 --- /dev/null +++ b/studies/ELF3TempEntrainment/protocols/TimeCourseExpressionAnalysis.txt @@ -0,0 +1,3 @@ +Timecourse expression Analysis: + +Seedlings were entrained in constant light (LL, 90 μmol m–2 s–1), under 12 h 22 °C:12 h 16 °C thermocycles for 8 d. On day 9, starting from ZT00, the samples were harvested every 4 h.All experiments were performed using three biological replicates. diff --git a/studies/ELF3TempEntrainment/protocols/WhiteLightImagingLD.txt b/studies/ELF3TempEntrainment/protocols/WhiteLightImagingLD.txt new file mode 100644 index 0000000000000000000000000000000000000000..11435cafe9a407447e9d6af03a871ca5aad2d884 --- /dev/null +++ b/studies/ELF3TempEntrainment/protocols/WhiteLightImagingLD.txt @@ -0,0 +1,5 @@ +White Light Imaging LD: + +Unless stated otherwise, seedlings were grown on vertically oriented plates in long days (LDs, 16 h light:8 h dark) with 90 μmol m–2 s-1 photosynthetically active radiation (PAR) using white fluorescent lamps (T5 4000K). Seedlings were grown at constant 16 °C or 22 °C for 8 d, or at constant 20 °C or 28 °C for 8 d. For temperature shift assays, seedlings grown at 20 °C for 4 d were shifted to 28 °C or were kept at 20 °C for an additional 4 d. + + diff --git a/studies/ELF3TempEntrainment/protocols/WhiteLightImagingSD.txt b/studies/ELF3TempEntrainment/protocols/WhiteLightImagingSD.txt new file mode 100644 index 0000000000000000000000000000000000000000..f827e0d52f766bd01be4c74e2b0eb26c23200a0e --- /dev/null +++ b/studies/ELF3TempEntrainment/protocols/WhiteLightImagingSD.txt @@ -0,0 +1,4 @@ +White Light Imaging SD: + +Unless stated otherwise, seedlings were grown on vertically oriented plates in short days (SDs, 8 h light:16 h dark) with 90 μmol m–2 s-1 photosynthetically active radiation (PAR) using white fluorescent lamps (T5 4000K). Seedlings were grown at constant 16 °C or 22 °C for 8 d, or at constant 20 °C or 28 °C for 8 d. For temperature shift assays, seedlings grown at 20 °C for 4 d were shifted to 28 °C or were kept at 20 °C for an additional 4 d. + diff --git a/studies/ELF3TempEntrainment/resources/PlantMaterial.txt b/studies/ELF3TempEntrainment/resources/PlantMaterial.txt new file mode 100644 index 0000000000000000000000000000000000000000..9772f3590d03951a711d591dd8c3d8f2d4dfe0bc --- /dev/null +++ b/studies/ELF3TempEntrainment/resources/PlantMaterial.txt @@ -0,0 +1,4 @@ +Plant Material: + +All A. thaliana lines used were in the Ws-2 or the Columbia-0 (Col-0) background. The elf3-4 (Zagotta et al., 1996; Hicks et al., 2001), gi-158 and elf3-4 gi-158 (Anwer et al., 2020), phyB-10 (Feldmann, 1991; Franklin et al., 2003), and pcl1-2 (Onai and Ishiura, 2005) null mutants in the Ws-2 background have been described previously. The elf3-4 phyB-10 was generating by crossing. The elf4-2, elf4-2 ELF3-OE, and elf3-1 ELF4-OE mutants in the Col-0 background have likewise been described previously (Nusinow et al., 2011; Box et al., 2015; Jung et al., 2020). Ws-2, elf3-4, phyB-10, and elf3-4 phyB-10 additionally harbor a CCR2:LUC reporter construct, and Ws-2 and pcl1-2 additonally harbor GI:LUC. +