title: GSM8865412: Rice-scATAC-Spikelet-s4-Rep2; Oryza sativa; ATAC-seq
library_construction_protocol: The nuclei isolation procedure was modified from the product information sheet of the CelLyticTM PN Isolation/Extraction Kit by Sigma. Briefly, the rice frozen organ was chopped in 1x NIBTA buffer (1 mM DL-Dithiothreitol solution, 1x ProtectRNA TM RNase Inhibitor, 1x cOmplete™, EDTA-free Protease Inhibitor Cocktail, 1x NIB, 0.3% Triton X-100) on pre-chilled plates. Transfer the homogenates to a new 15 mL tube. The tube was shaken for 5 min on ice. The lysate was transferred into a 30-μm Smartstrainer and collected using a new 15 mL tube. Spin the 15 mL tube for 10 min at 1,260 × g. Decant the supernatant and resuspend the pellet in 4 mL of 1x NIBTA buffer. Put 5 mL of 75% PercoII® solution (3.75 mL PercoII and 1.25 mL NIBTA buffer) in a new 15 mL tube and carefully transfer the lysate on top of the Percoll® layer. Centrifuge for 30 min at 650 × g. Most of the nuclei are banded at the interface between the 1x NIBTA buffer and the PercoII® layers. Gently collect the nuclei band in a new 15 mL tube, and 1x NIBTA buffer was added to 10 mL. Centrifuge for 5 min at 1260 × g. Wash the nucleus pellet twice with 1x NIBTA buffer. Nuclei were resuspended in diluted nuclei buffer (10x Genomics) to a final concentration of 4,000 nuclei per µL and used as input for single-cell multiome ATAC + gene expression and scRNA-seq library preparation (5 µL; approximately 20,000 nuclei). A total of 20,000 nuclei from each rice sample were pooled for single-cell multiome library generation using the 10X genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit. The nuclei were counted by Luna FL (Logos Biosystems). GEMs were generated using the 10X Chromium Controller with one lane of GEM chip (Chromium Next GEM Chip J) for each sample. scATAC and gene expression libraries from each pool were generated Glbizzia Biosciences following the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide. In brief, nuclei were incubated in a transposition mix that includes transposases that enter the nuclei and fragment DNA in open regions of chromatin, while simultaneously adaptor sequences are added to the fragments. Single Cell Multiome ATAC + GEX Gel Beads capture the adaptor sequences on DNA fragments as well as the poly-A tail of mRNA and GEMs are generated and incubated, resulting in 10x barcoded DNA from the transposed DNA (for ATAC) and 10x barcoded, full-length cDNA from poly-adenylated mRNA (for GEX). cDNA was then purified, amplified and sequenced per the manufacturer's recommendations (10x Genomics). The libraries were sequenced with Illumina NovaSeq 6000 under the modes of PE150 (scRNA-seq library) and PE50 (scATAC-seq library).