GSM8383560: Wheat root, root cap region; Triticum aestivum; OTHER
Roots were harvested fresh and embedded in pre-cooled OCT (Sakura) and stored at -80 °C until processed. The pre-frozen root tissues in OCT were cross-sectioned at 10 μm thickness using a Leica CM1950 cryostat. All following steps were performed according to the protocols from (Chen et al., 2022; Xia et al., 2022) with small modifications.  Stereo-seq chip was pretreated with 0.01% poly-lysine for 10 minutes at room temperature before use. Tissue sections were adhered to the Stereo-seq chip surface and incubated on a slide warmer at 37 °C for 8 minutes. Then, tissues were fixed in methanol and incubated at -20 °C for 30 minutes and afterwards stained with Fluorescent Brightener 28 (FB) and Qubit ssDNA. Imaging was performed with a Motic fluorescence microscope. Then the tissue sections were permeabilized at 37 °C for 12 minutes followed by a washing step with 0.1x SSC buffer (Thermo, AM9770) containing 0.05 U/μL RNase inhibitor (NEB, M0314L). RNA was reverse transcribed for 3 hours at 42 °C, tissue sections were washed twice with 0.1x SSC buffer and digested with Tissue Removal buffer at 55 °C for 10 minutes. cDNA release mix was added and the cDNA was released during an overnight treatment  at 55 °C. cDNA was purified using DNA Cleanup Beads AMPure(R)  XP (Agencourt), and amplified using PCR. A total of 30 ng of DNA were used for fragmentation, performed at 55 °C for 10 minutes. Fragmentation products were amplified using PCR,  purified using the DNA Cleanup Beads AMPure(R)  XP, and sequenced (paired-end 100 bp) on an MGI-DNBSEQ-Tx sequencer.