title: GSM9261294: ZmRap2.7, tsCUT&Tag, Rep2; Zea mays; OTHER
library_name: GSM9261294
library_construction_protocol: For bulk RNA-seq, SAM samples at 14, 16, 18, 22, 26, and 28 DAS were flash-frozen with liquid nitrogen immediately after collection. Total RNA was extracted using the Direct-zol RNA Microprep Kit (Zymo Research). For ATAC-seq, approximately 25 freshly dissected SAMs were digested in 15 μl protoplast lysis buffer at the bottom of 1.5 ml tube for 2 h at room temperature in the dark with gentle shaking. For scRNA-seq, approximately ~300 SAMs with two youngest leaf primordia were dissected and enzymatically digested in 50 μl protoplast lysis buffer at the bottom of 2 ml tube for 2 h at room temperature in the dark with gentle shaking to release protoplasts. After digestion, the reaction was stopped by dilution using 1.5 ml protoplast washing buffer (lysis buffer without enzyme) and filtered through 30-μm strainers to remove debris and large aggregates. The filtrate was then centrifuged at 300 g for 3 min at room temperature to collect protoplasts. Pellets were gently resuspended and washed twice with protoplast washing buffer under the same centrifugation conditions. After washing, a final filtration step was performed to remove cell aggregates and obtained a suspension of single, dissociated cells. Cell concentration and viability were assessed by fluorescein diacetate (FDA) staining using a hemocytometer. Only samples with ≥ 75% viable cells were used. For bulk RNA-seq, sequencing libraries were constructed with the MGIEasy RNA Library Prep Kit according to the manufacturer's instructions. Nuclei were released by adding 1 ml release buffer (1 × PBS, 1 × protease inhibitor, 0.3% Triton X-100) and gentle pipetting. For ATAC-seq, nuclei were then collected by centrifugation and resuspended in 20 μl 1 × TTBL buffer (Vazyme, TD501). Nuclei integrity and concentration were assessed by DAPI staining. Approximately 5,000 nuclei were subjected to Tn5 transposition (Vazyme, TD501) in the presence of 0.3% Triton X-100 at 37 °C for 30 min. Transposed DNA was immediately purified using Qiagen MinElute kits and amplified for 10-13 PCR cycles according to the manufacturer's instructions (VAHTS, TD501). Amplified DNA was size-selected using KAPA DNA Clean Beads (150-650 bp) to generate the final library, which was sequenced on an Illumina HiSeq X Ten platform (paired-end, 2 × 150 bp). For  tsCUT&Tag, full-length coding sequences of ZmMADS69 and ZmRap2.7 were cloned into the PM999-GFP vector, and plasmids were purified to transfection grade. Protoplasts were prepared from etiolated B73 seedlings grown in the dark and transfected with the GFP-tagged constructs using PEG-mediated transformation. After overnight incubation, protoplasts were processed using the Hyperactive In-Situ ChIP Library Prep Kit (Vazyme, Nanjing, China) according to the manufacturer's instructions. Chromatin was immunoprecipitated with anti-GFP antibody (1:100 dilution), and tagmented DNA fragments were PCR-amplified and size-selected to generate sequencing libraries. Libraries were subjected to paired-end sequencing (2 × 150 bp) on an Illumina NovaSeq 6000 platform. For scRNA-seq, approximately 15,000 protoplasts were loaded into the BD Rhapsody™ Single-Cell Capture system (BD Biosciences) for scRNA-seq library construction according to the manufacturer's protocol. Libraries were sequenced on an Illumina NovaSeq 6000 platform with paired-end 150 bp reads.