protocols added in Studies and Assays

assays/ClockGating/protocols/RNAExtractionCDNASynthesis.txt

+
+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

assays/qRTexpressionTimecourse/protocols/RNAExtractionCDNASynthesis.txt

+
+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

studies/ELF3TempEntrainment/protocols/PlantMaterialGrowthConditions.txt

+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.