title: GSM6761325: ge10_24h_section_A; Hordeum vulgare; RNA-Seq
library_construction_protocol: Seeds were harvested at 0, 1, 3, 6 and 24 HAI. The seeds for the Visium experiment were collected at the indicated time-points and hand-dissected longitudinally. The seeds were immediately snap frozen in an isopentane bath (2-methylbutane, Sigma Aldrich, cat no. 270342-1L) for 1 min, subsequently embedded in Optimal Cutting Temperature (OCT, Tissue-Tek, cat no. 4583) and the blocks frozen in the isopentane bath and kept on dry-ice. The cryoblocks were cut on a cryostat (Leica Biosystems; MA, USA) to a thickness of 8 µm at -18°C. The sections were carefully place on a prechilled Tissue Optimization (TO) or Gene Expression (GE) slide and kept frozen at -80 °C until required. The slides were dried on a metal plate (PN-1000317, 10X Genomics) in the thermocycler at 37 °C for 1 min, fixed with chilled methanol at -20°C for 30 min, stained with 0.1% (wt/vol) Safranin O (Sigma-Aldrich, cat no. S8884-25G) in 50% (vol/vol) EtOH with 2U/μl RNase inhibitor at room temperature (RT) for 5 min and washed in increasing concentrations of EtOH until the discard liquid was clear. After drying at 37°C for 1 min, the slides were mounted in 85% (vol/vol) glycerol + 2U/μl RNase inhibitor, covered with a coverslip and images of sections were taken using Axio-Imager.M2 microscope (Zeiss). TO slides were fully recorded in one file meanwhile GE capture areas were imaged individually. Raw images were stitched together using Zen blue software v2.5 (Zeiss). Prior to imaging, the microscope settings were validated using the Visium Imaging Test Slide (PN-2000235). The slide contained the same 8 areas with fiducial frames that the TO slide. However, the test slide only contained 4 areas coated with fluorescent spots to set the boundaries of the capture areas, the focus, and the fluorescence conditions. The optimised settings were saved into the TO macro for following experiments. After imaging, the coverslip was removed immediately immersing the slide at 45° angle in 3x SSC buffer with the coverslip surface completely submerged and facing down. The coverslip slowly separated from the slide without damaging the samples nor the capture areas. The slide was immersed again in 3x SSC buffer to fully remove the mounting media and was let dry for a few seconds. To pre-permeabilize the tissue, the slides were mounted in a plastic cassette and sections incubated in pre-permeabilization solution (48 μl Exonuclease I buffer, NEB, cat no. B0293S; 4.5 µl of BSA, Sigma-Aldrich, cat no. A7039-100G; and 2% (w/v) PVP40, Sigma-Aldrich, cat no. PVP40-1KG) (PMID: 30353173) at 37°C for 30 min. This was followed with a wash with 0.1× SSC buffer (Sigma-Aldrich, cat no. S6639L). The sections were permeabilized with Permeabilization mix™ (10X Genomics) at 37°C for different times (1, 3, 6, 15, 30 min TO slides) or for 3 min (GE slides). Then, wells were washed with 0.1× SSC buffer. After permeabilization, Reverse transcription mixture™ (10X Genomics) was added to each section and incubated at 56°C for 45 min as described in the 10X Genomics User guide (PN-1000186, CG000239_VisiumSpatialGeneExpression_UserGuide_RevD).  To remove the tissue, a hydrolytic enzyme mixture was prepared by adding 70 µL of each enzyme; cellulase (Yakult -"ONOZUKA" R-10, cat no. YAKL0012), pectate lyase (Megazyme, cat no. E-PCLYAN2), xyloglucanase (Megazyme, cat no. E-XEGP), endo 1,4 β-xylanase (Megazyme, cat no. E-XYNACJ), endo 1,4 β-mannanase (Megazyme, cat no. E-BMACJ) and lichenase (Megazyme, cat no. E-LICHN) to 140 µl of 250 mM sodium citrate buffer (Sigma-Aldrich, cat. no. 71497-250 G), pH 6.6. The enzymatic mixture was added to the wells and incubated in a Thermo Mixer at 37 °C for 90 min with shaking (300 r.p.m.). The wells were washed with 0.1x SSC buffer.  Samples were incubated with 10% (v/v) Triton X-100 solution in a Thermo Mixer at 56 °C for 1 h with shaking (300 r.p.m.), followed by a wash with 0.1x SSC buffer. Next wash consisted in a mixture of RLT buffer (Qiagen ref.79216) with 1% β-mercaptoethanol, which was incubated in a Thermo Mixer at 56 °C for 1 h with shaking (300 r.p.m.) and followed by a wash with 0.1x SSC buffer. A final incubation with 70 µl proteinase K mixture (60 µl of proteinase K (Qiagen, cat no. 19131) and 420 µl of PKD buffer (Qiagen, cat no. 1034963) was performed in a Thermo Mixer at 56 °C for 1 h with shaking (300 r.p.m.).  Hybridization chamber was detached, and the slide washed in a petri dish with 50 °C pre-warmed wash buffer 1 (2× SSC/0.1% SDS) at 50 °C for 10 min with shaking (300 rpm). The slides were further washed with wash buffer 2 (0.2× SSC) and wash buffer 3 (0.1× SSC) at RT for 1 min with shaking (300 rpm). The slide was spin-dried in a swing-bucket centrifuge. Following the Gene Expression workflow, libraries from the different capture areas were synthesized as described in 10X User guide (PN-1000190, CG000239_VisiumSpatialGeneExpression_UserGuide_RevD). Visium libraries were sequenced on an Illumina NextSeq 550 platform according to the 10X Genomics Visium manufacturer's instructions (PN – XXXXXXXXXX, CG000239_VisiumSpatialGeneExpression_UserGuide_RevD), targeting 100 million reads using dual indexing kit TT set A (PN-1000215, 10X Genomics). 10x Genomics Visium gene expression