ERX1066760 E-MTAB-3830:Sample 13 E-MTAB-3830:Sample 13 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813889 SAMEA3506740 E-MTAB-3830:Sample 13 Sample 13 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: mature developmental stage Experimental Factor: none stimulus ERX1066761 E-MTAB-3830:Sample 14 E-MTAB-3830:Sample 14 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813890 SAMEA3506741 E-MTAB-3830:Sample 14 Sample 14 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: mature developmental stage Experimental Factor: none stimulus ERX1066762 E-MTAB-3830:Sample 15 E-MTAB-3830:Sample 15 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813891 SAMEA3506742 E-MTAB-3830:Sample 15 Sample 15 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: mature developmental stage Experimental Factor: none stimulus ERX1066763 E-MTAB-3830:Sample 7 E-MTAB-3830:Sample 7 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813892 SAMEA3506743 E-MTAB-3830:Sample 7 Sample 7 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: post-meiotic developmental stage Experimental Factor: none stimulus ERX1066764 E-MTAB-3830:Sample 8 E-MTAB-3830:Sample 8 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813893 SAMEA3506744 E-MTAB-3830:Sample 8 Sample 8 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: post-meiotic developmental stage Experimental Factor: none stimulus ERX1066765 E-MTAB-3830:Sample 9 E-MTAB-3830:Sample 9 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813894 SAMEA3506745 E-MTAB-3830:Sample 9 Sample 9 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: post-meiotic developmental stage Experimental Factor: none stimulus ERX1066766 E-MTAB-3830:Sample 1 E-MTAB-3830:Sample 1 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813895 SAMEA3506746 E-MTAB-3830:Sample 1 Sample 1 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: tetrad developmental stage Experimental Factor: none stimulus ERX1066767 E-MTAB-3830:Sample 2 E-MTAB-3830:Sample 2 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813896 SAMEA3506747 E-MTAB-3830:Sample 2 Sample 2 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: tetrad developmental stage Experimental Factor: none stimulus ERX1066768 E-MTAB-3830:Sample 3 E-MTAB-3830:Sample 3 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813897 SAMEA3506748 E-MTAB-3830:Sample 3 Sample 3 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: tetrad developmental stage Experimental Factor: none stimulus ERX1066769 E-MTAB-3830:Sample 16 E-MTAB-3830:Sample 16 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813898 SAMEA3506749 E-MTAB-3830:Sample 16 Sample 16 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: mature developmental stage Experimental Factor: heat stress stimulus ERX1066770 E-MTAB-3830:Sample 17 E-MTAB-3830:Sample 17 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813899 SAMEA3506750 E-MTAB-3830:Sample 17 Sample 17 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: mature developmental stage Experimental Factor: heat stress stimulus ERX1066771 E-MTAB-3830:Sample 18 E-MTAB-3830:Sample 18 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813900 SAMEA3506751 E-MTAB-3830:Sample 18 Sample 18 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: mature developmental stage Experimental Factor: heat stress stimulus ERX1066772 E-MTAB-3830:Sample 10 E-MTAB-3830:Sample 10 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813901 SAMEA3506752 E-MTAB-3830:Sample 10 Sample 10 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: post-meiotic developmental stage Experimental Factor: heat stress stimulus ERX1066773 E-MTAB-3830:Sample 11 E-MTAB-3830:Sample 11 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813902 SAMEA3506753 E-MTAB-3830:Sample 11 Sample 11 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: post-meiotic developmental stage Experimental Factor: heat stress stimulus ERX1066774 E-MTAB-3830:Sample 12 E-MTAB-3830:Sample 12 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813903 SAMEA3506754 E-MTAB-3830:Sample 12 Sample 12 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: post-meiotic developmental stage Experimental Factor: heat stress stimulus ERX1066775 E-MTAB-3830:Sample 4 E-MTAB-3830:Sample 4 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813904 SAMEA3506755 E-MTAB-3830:Sample 4 Sample 4 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: tetrad developmental stage Experimental Factor: heat stress stimulus ERX1066776 E-MTAB-3830:Sample 5 E-MTAB-3830:Sample 5 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813905 SAMEA3506756 E-MTAB-3830:Sample 5 Sample 5 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: tetrad developmental stage Experimental Factor: heat stress stimulus ERX1066777 E-MTAB-3830:Sample 6 E-MTAB-3830:Sample 6 ERP011644 E-MTAB-3830 Small noncoding RNAs in tomato pollen ERS813906 SAMEA3506757 E-MTAB-3830:Sample 6 Sample 6 ncRNA-Seq TRANSCRIPTOMIC RANDOM The short-term heat stress treatment and collection of pollen samples were performed according to the protocol developed by the SPOT-ITN consortium. For heat treatment plants were transferred in a preheated growth chamber and exposed to 38oC for 1 h (Figure S 1) under artificial light (Photosynthetically active radiation (PAR) = 85,927±4,7 μmol photons m−2s−1). The temperature was then gradually decreased to 25oC within 30 minutes and plants were allowed to recover for an additional hour at 25oC. Untreated plants were kept in the growth chamber for the same time period at control temperature of 25oC. In a preliminary experiment (data not shown) the tomato flower bud size has been correlated with three stages of pollen development, using DAPI (4, 6-diamidino-2-phenylindole) staining as shown by Chaturvedi et al. (38). Flower material was harvested 1.5 h after HS and immediately collected in the containers kept on ice. Control samples were harvested from a parallel set of non‐stressed plants while performing the HS treatments. Flower buds were sorted according to the corresponding stages as follow: flower buds of 4‐6 mm in length for tetrad (T) (meiotic stage/microspore mother cell), 6‐8 mm in length for post-meiotic (PM) stage (microspores), and =/> 10 mm in length for mature (M) (bicellular pollen) pollen grains (Figure S 1). Sepals from the flower buds of tetrad stage and sepals and petals from the flower buds of post‐meiotic and mature stages were gently removed using forceps. Additionally stigma and the style from the flower buds of post‐meiotic and mature stages were excised in order to avoid the contamination by parts of the gynoecium during pollen preparation. Immediately after removing aforementioned parts of the flower buds, released anthers were immersed in germination solution [KNO3 (1 mM); Ca(NO3)2 (3 mM); MgSO4 (0,8 mM), H3BO3 (1,6 mM)] kept on ice prior pollen isolation and purification. Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. To ensure that the material was free of contaminants from other anther tissues, isolated pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100g. The supernatant was discarded, while pollen cell pellet re‐suspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C. The supernatant was discarded and pollen samples were stored in the liquid nitrogen. Prior to first centrifugation the pollen stage and purity were confirmed by staining with 1 μg mL-1 DAPI and visualized under a fluorescent microscope [38] (Figure S 1). Control and heat-stressed pollen samples were collected from three independent experiments performed during three consecutive days. Samples derived from one day were treated as biological replica. Growth conditions included a 12 hour light-dark cycle with a day temperature of 24-26°C and night temperature of 18-20°C, and relative humidity at 65-70%. The daily solar radiation in the greenhouse was supplemented to at least 190 μmol photons m-2 s-1 from a series of high-pressure sodium lamps 400 W SON-T (Philips Electronics N.V. Amsterdam, Netherlands) after the appearance of the first inflorescence. During the first 6 weeks of development, plants were watered every two days and subsequently every day. RNA was isolated using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. sncRNA libraries were prepared using the proprietary TrueQuant method for elimination of PCR bias [39]. Briefly, adapters were ligated to the 3’- and 5’-ends to total RNA by T4 RNA ligase, reverse transcribed and PCR amplified. The final PCR product was size-selected on a PAA-gel . Illumina HiSeq 2000 Experimental Factor: tetrad developmental stage Experimental Factor: heat stress stimulus