Illumina NovaSeq 6000 paired end sequencing; Marchantia abiotic stress combination and diurnal gene expression atlas
Marchantia abiotic stress combination and diurnal gene expression atlas
Sample 8_p
In order to determine the ideal stress condition to be used for cross-stress experiments, a series of severity was used for heat, cold, salt, osmotic, light, dark and nitrogen deficiency stresses. For each variation of stress, three agar plates containing 9 gemmae each were plated, where two plates were used as material for RNA sequencing and one plate was kept for observation until 21 days after plating (DAP).  For salt, osmotic, and nitrogen deficiency stress, the gemmae were plated on half-strength Gamborg B-5 Basal agar (pH 5.5, 1.4% agar, 24.7 mM KNO3) supplemented with 20 - 200 mM NaCl (20 mM steps), 50-400 mM mannitol (50 mM steps) and KNO3 concentration ranging from 90 to 0% (22.3 - 0 mM, 2.5 mM steps), respectively. The potassium concentration in nitrogen deficiency agar was maintained using equimolar concentrations of KCl. Gemmae subjected to heat, cold, light and dark stresses were plated on normal half-strength Gamborg B5 agar (pH 5.5, 1.4% agar, 24.7 mM KNO3).  On day 15, whole plants were harvested by pooling 3 plants into 2 mL Eppendorf tubes, flash frozen in liquid nitrogen and stored at -80oC. This was done in triplicates, resulting in 3 (replicates) * 3 (pooled plants) used for each RNA-seq sample.  Male Marchantia polymorpha, accession Takaragaike-1 (kindly provided by Prof. Chen Zhong from National Institute of Education, Singapore) was propagated on half-strength Gamborg B-5 Basal agar (1% sucrose, pH 5.5, 1.4% agar) in deep well plates (SPL Biosciences, SPL 310101) at 24oC under continuous LED light at 60 Em-2s-1. For salt, osmotic, and nitrogen deficiency stress, the gemmae were plated on half-strength Gamborg B-5 Basal agar (pH 5.5, 1.4% agar, 24.7 mM KNO3) supplemented with 20 - 200 mM NaCl (20 mM steps), 50-400 mM mannitol (50 mM steps) and KNO3 concentration ranging from 90 to 0% (22.3 - 0 mM, 2.5 mM steps), respectively. The potassium concentration in nitrogen deficiency agar was maintained using equimolar concentrations of KCl. Gemmae subjected to heat, cold, light and dark stresses were plated on normal half-strength Gamborg B5 agar (pH 5.5, 1.4% agar, 24.7 mM KNO3).Plates were grown at 24oC under continuous LED light at 60 Em-2s-1 from days 0 to 13. For dark stress, plates were moved to the plant growth chamber (HiPoint M-313) on days 8, 9, 10, 11, 12, 13 and 14 for growth in darkness at 24oC for 7, 6, 5, 4, 3, 2 and 1 day(s), respectively. On day 14, all plates were transferred to the plant growth chamber (HiPoint M-313) for 24 hours. Control and plates for salt, osmotic and nitrogen deficiency stresses were maintained at 24oC  under continuous LED light at 60 Em-2s-1 in the plant growth chamber. For heat, cold, dark and light stresses, normal growth conditions were used with the following modifications. For heat and cold stress, 27oC to 36oC (3oC steps) and 3oC to 12oC (3oC steps) were used, respectively. For light stress, plants were subjected to 20% to 100% chamber capacity of light output (115 to 535 Em-2s-1), at steps of 20%, approximating to 100 Em-2s-1 per step.For diurnal gene expression, three agar plates containing 9 gemmae each were plated for each sampling time point half-strength Gamborg B-5 Basal agar (0% sucrose, pH 5.5, 1.4% agar). The plants were grown at 24oC under continuous LED light at 60 Em-2s-1. On day 8, plates were transferred to the plant growth chamber and grown at 24oC under LED light at 60 Em-2s-1 with a 12 h/12 h day/night cycle. On day 15, plants were harvested at Zeitgeber time (ZT) 2, 6, 10, 14, 18 and 22. The plants were stored in 2 mL Eppendorf tubes (3 plants per tube) and flash frozen in liquid nitrogen before storage at -80oC. Plants from each Eppendorf tube were ground into fine powder in liquid nitrogen using mortar and pestle. RNA was extracted using the SpectrumTM Plant Total RNA Kit (Sigma, STRN-250) using Protocol A (750 L Binding Solution) according to manufacturers instruction with on-column DNase digestion using 60 L of DNase mixture (15 L RQ1 RNase-Free DNase (Promega, M6101), 6 L RQ1 DNase 10X Reaction Buffer and 19 L nuclease free water) per column.  Library construction from total RNA including eukaryotic mRNA enrichment by oligo(dT) beads, library size selection and PCR enrichment was performed by Novogene. Libraries were constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina kit.